Peptide, peptide analog and amino acid analog protease inhibitors

ABSTRACT

Methods of use of compounds and compounds for the treatment of disorders characterized by the cerebral deposition of amyloid are provided. Among the compounds are those of formulae (I), (II) and (III): ##STR1## in which R 1  is preferably 2-methyl propene, 2-butene, norleucine; R 2 , R 4 , and R 8  are each independently methyl or ethyl; R 3  is preferably iso-butyl or phenyl; R 5  is preferably iso-butyl; R 6  is H or methyl; R 7  --(Q) n  is preferably benzyloxycarbonyl or acetyl; Q is preferably --C(O)--; R B  is preferbly iso-butyl; R A  =--(T) m  --(D) m  --R 1 , is which T is preferably oxygen or carbon, and D is preferably a mono-unsaturated C 3-4  alkenyl being more preferred; and X is an alcohol, particularly a secondary alcohol.

This is a continuation of pending application Ser. No. 08/403,420 toMunoz et al., filed Mar. 13, 1995, which is a continuation-in-part ofpending application Ser. No. 08/369,422, filed Jan. 6, 1995, which is acontinuation-in-part of U.S. application Ser. No. 08/369,422, now U.S.Pat. No. 5,804,560 to McDonald et al., filed Jan. 6, 1995, entitledPEPTIDE AND PEPTIDE ANALOG PROTEASE INHIBITORS. The subject matter ofU.S. application Ser. No. 08/369,422, now U.S. Pat. No. 5,804,560, andis herein incorporated in its entirety by reference.

FIELD OF THE INVENTION

This invention relates to peptidyl compounds useful for a variety ofphysiological end-use applications. More specifically, peptide and aminoacid analogs that are protease inhibitors are provided. These inhibitorsare useful in the treatment of certain diseases, includingneurodegenerative diseases characterized by the accumulation of amyloidplaques, and in diseases characterized by degeneration of the neuronalcytoskeleton.

BACKGROUND OF INVENTION

Proteases play a key role in metabolism and in the pathology of numerousdisorders. As a consequence, compounds that specifically inhibitprotease activity are often therapeutic. For example, renin is anaspartyl protease that cleaves angiotensinogen to angiotensin I.Angiotensin I is hydrolyzed by angiotensin converting enzyme (ACE) toangiotensin II, which is a potent vasoconstrictor and stimulant ofaldosterone secretion. Compounds that inhibit the activity of ACE, suchas captopril an orally active ACE inhibitor, are therapeuticallyeffective for treating hypertension and congestive heart failure. Renininhibitors are thought to have similarly beneficial therapeuticactivity.

Activated ketone-based inhibitors have found uses as inhibitors of fourdifferent classes of proteases, serine proteases, aspartyl proteases,cysteine proteases and metalloproteases, because they exist as hydratesin aqueous media and directly serve as transition state analogs and/orreact with a nucleophilic residue (such as the serine hydroxyl orcysteine sulfhydryl) to form a reversible hemiacetal-type intermediate.For example, phenylalkyl ketones are potent and competitive reversibleinhibitors of interleukin 1-β converting enzyme (ICE) and thus, arethought to have therapeutic use for treatment of certain chronicinflammatory disease states. The precursor alcohols for these ketoneshave been reported to have substantially reduced protease activitycompared to the corresponding ketone [see, e.g., Patel et al. (1988)Tetrahed. Lttrs. 29:4665-4668; Patel et al. (1993) J. Med. Chem.36:24310-2447].

Trifluoromethyl ketones are inhibitors of, for example, human leukocyteelastase (HLE), which is a serine protease. This enzyme has beenimplicated as a pathogenic agent in a variety of disorders, includingpulmonary emphysema, rheumatoid arthritis, adult respiratory distresssyndrome (ARDS), glomerulonephritis and cystic fibrosis [see, e.g.,Skiles et al. (1992) J. Med. Chem. 35:641-662; Angelastro et al. (1994)J. Med. Chem. 37:4538-4554].

Proteases are also implicated in diseases, such as Alzheimer's Disease(AD), that are characterized by the accumulation of amyloid plaques.Amyloidogenic Aβ peptides (Aβ) are the principle component of theamyloid plaques that accumulate intracellularly and extracellularly inthe neuritic plaques in the brain in AD. Aβ is a 4.5 kD protein, about40-42 amino acids long, that is derived from the C-terminus of amyloidprecursor protein (APP). APP is a membrane-spanning glycoprotein that,in the normal processing pathway, is cleaved inside the Aβ protein toproduce α-sAPP, a secreted form of APP. Formation of α-sAPP precludesformation of Aβ. It has been proposed that Aβ accumulates by virtue ofabnormal processing of APP, so that compounds that inhibit the activityof enzymes responsible for Aβ production are being sought [see, e.g.,Wagner et al. Biotech. Report (1994/1995), pp.106-107; and Selkoe (1993)TINS 16:403-409].

Because proteases are implicated in numerous disorders, there is a needto develop potent and specific inhibitors of these enzymes. Therefore,it is an object herein to provide methods of treating disorders in whichprotease activity plays a pathological role. It is also an object hereinto provide protease inhibitors.

SUMMARY OF THE INVENTION

Methods of inhibiting proteases are provided. The protease inhibitorsare peptidyl, peptidyl analog and amino acid analog alcohols,particularly haloalkyl secondary alcohols. These inhibitors include thecorresponding alcohols of any peptidyl or peptidyl analog ketones oraldehydes that inhibit proteases in cell-free assays [see, EP 0 410 411A2, which is based on U.S. application Ser. No. 07/385,624, WO 92/20357,which is based on U.S. application Ser. No. 07/704,449, EP 0 364 344 A2,which is based on U.S. application Ser. No. 07/254,762]. Peptidyl,peptidyl analog and amino acid analog alcohol protease inhibitors thatdo not correspond to such known ketone or aldehyde inhibitors are alsoprovided herein.

Thus, di- and tri-peptide analogs and amino acid analogs and methods oftreating certain disorders, particularly cognitive disorders, but anydisorder in which a protease, particularly a serine, cysteine oraspartyl protease, is involved in the pathology, and methods ofinhibiting proteases using the compounds are provided. Among thecompounds used in the methods are peptidyl or peptidyl analog haloalkylalcohols, particularly secondary alcohols and fluoro-lower-alkylalcohols.

In particular, the methods, particularly the methods for treatingcognitive disorders involving accumulation of amyloid plaques in thebrain tissue, use compounds having formulae: ##STR2## or the hydratesand isosteres, diastereomeric isomers and mixtures thereof, orpharmaceutically acceptable salts thereof.

X has the formula: ##STR3## where A and B are independently selectedfrom among H, halogen, alkyl, heterocycle, arylalkyl, haloalkyl, inwhich the alkyl groups are straight or branched chains or form a fusedring(s) or preferably a single ring, aryl, particularly halo-substitutedaryl, alkylhaloaryl, (CH₂)_(r) CHN₂, CH₂ (CH₂)_(r) OR_(D), CH₂ (CH₂)_(r)OZ_(D), --(CH₂)_(r+1) W, --(CH₂)_(r+1) U in which:

the carbon or heterocyclic ring(s) contain from 3 to about 20 members,preferably 5-7, in the ring(s), which are unsubstituted or aresubstituted with one or more substituents independently selected from G,

the aryl, cyclic and heterocyclic portions of X can be furthersubstituted with G;

r is 0-5, preferably 0-3, more preferably 0 or 1, most preferably 0;

G is halogen, preferably F, lower alkyl, alkoxy, OH, haloalkyl,preferably CF₃, NO₂, nitrile, S-alkyl, phenyl, and --NRR;

R is H or alkyl, preferably lower alkyl, OH and halo-lower alkyl,particularly CF₃ ; the aryl groups preferably contain from 5-6 membersand are unsubstituted or substituted with one or more substituentsindependently selected from G, which is preferably halogen, morepreferably fluoro;

the heterocyclic rings preferably contain one or two heteroatoms andpreferably contain 5 or 6 members;

Z_(D) is haloalkyl, in which the alkyl portion is straight or branched,cyclic, or mixtures thereof, the straight or branched chains containfrom 1 to about 10, preferably 1-8, more preferably 1-6, most preferably1-3, carbons in the chain, and the cyclic portions contain from 3 toabout 10, preferably 3-7, carbons in the cycle, and the halo portion ispreferably fluoro;

U is --OR_(D) or --NR_(D) R_(D) ;

R_(D) is selected from among H, alkyl, preferably lower alkyl, morepreferably C₁₋₄ alkyl, phenyl, and phenethyl;

W is --OR_(D), --SR_(D), and --NR_(D) R_(D), or a heterocyclic moiety,such as a thiazolyl, preferably containing 4-6, more preferably 5 or 6members in the ring, and preferably one or two heteroatoms, selectedfrom O, S, or N, in the ring. Preferably, at least one of A or B is H.

In particular, X is selected from among (CH₂)_(r) CH(OH)halo-substitutedalkyl, preferably --CH(OH)C_(k) H.sub.(2k+1-s) F_(s) in which k is 1-6,preferably 1-3, s is 0 to 2k+1; --CH(OH)C₆ H.sub.(5-q) F_(q) in which qis 0 to 5; --(CH₂)_(r) CH(OH)CF₃, --(CH₂)_(r) CH(OH)C₂ F₅, --(CH₂)_(r)CH(OH)H, --(CH₂)_(r) CH(OH)(CH₂)_(r) CHN₂, --(CH₂)_(r) CH(OH)haloalkyl,--(CH₂)_(r) CH(OH)(CH₂)_(r) C(O)U, --(CH₂)_(r) CH(OH)(CH₂)_(r) U,--(CH₂)_(r) CH(OH)CH₂ W and --C(halo)₂ aryl. X is more preferably--CH(OH)CF₃ or --CH(OH)C₂ F₅. In all embodiments, halo is preferablyfluoro.

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(A), R_(B), Q and n are selected fromamong (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii) as follows:

(i) R₁, R₃, R₅, and R_(B), are each independently selected from a sidechain of a naturally occurring α-amino acid, H, alkyl, preferably lower(C₁₋₆) alkyl, alkenyl, preferably C₂₋₁₀ alkenyl, alkynyl, preferablyC₂₋₆ alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl,heteroaralkyl, heteroaralkenyl, Y-substituted aryl, aralkyl, aralkenyl,aralkynyl, and Z-substituted heteroaryl, heteroaralkyl, heteroaralkenyl,in which Y is selected from halogen, lower alkyl, alkoxy, OH, haloalkyl,preferably CF₃, NO₂, nitrile, S-alkyl, phenyl, and --NRR, R is H oralkyl, preferably lower alkyl, OH and halo-lower alkyl, particularlyCF₃, Z is lower alkyl, preferably C₁₋₄ alkyl, or halo lower alkyl,preferably C₁₋₄ haloalkyl, more preferably CF₃ ;

R₂, R₄, R₆, and R₈ are each independently selected from among H andlower alkyl, preferably C₁₋₄ alkyl;

R₇ is selected from among C₁₋₆ alkyl, aryl, alkenyl, 9-fluoroenyl,aralkyl, aralkenyl, aralkynyl the aryl groups are unsubstituted or aresubstituted with Z;

Q is selected from among --C(O)--, --O--C(O), --S(O)₂ -- and HN--C(O)--;

n is zero or one;

R_(A) is --(T)_(m) --(D)_(m) --R in which T is O or NH, and D is C₁₋₄alkyl or C₂₋₄ alkene; and m is zero or one; or

(ii) R₁, R₂, R₃, R₄, R₅, R₈ and Y are selected as in (i), (iv) or (v);

V is OH, halogen, lower alkyl, preferably methyl or ethyl orhalogen-substituted lower alkyl, preferably methyl or ethyl, and ispreferably preferably OH;

n is zero; and

R₆ and R₇ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₃ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₃ alkyl, with the proviso that there is atleast one carbon atom between the N to which R₆ and R₇ are each attachedand the heteroatom; and

(b) R₆ and R₇ are unsubstituted or substituted with one or moresubstituents selected from Y or preferably V, which is selected from OH,halogen, lower alkyl, preferably methyl or ethyl, or halogen-substitutedlower alkyl, preferably methyl or ethyl, and is more preferably OH; and

(c) together with the atoms to which each is attached form aheterocyclic moiety, preferably a 5-6 atomed heterocyclic moiety, morepreferably selected from among morpholino, thiomorpholino, pyrrolidinyl,or V-substituted pyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(iii) R₁, R₂, R₃, R₄, R₅, R₈, R_(A), R_(B) are selected as in (i);

V is as defined in (ii);

Q is C(O);

n is one; and

R₆ and R₇ are each independently selected as follows:

(a) from carbonyl (C═O), phenyl, a heteroatom, lower alkyl, preferablyC₁₋₃ alkyl, or lower alkyl linked to a heteroatom, preferably C₁₋₃alkyl, and

(b) each is unsubstituted or substituted with Y, preferably with V, and

(c) together with the atoms to which they are attached form a cyclicmoiety, preferably a 4-6 membered cyclic or 8-12 bicylic moiety, and

(d) and R₆ and R₇ are selected with the proviso that when two or moreheteroatoms are present there is a carbon atom between the heteroatoms;and

(e) when the moiety is a heterocycle, it is preferably succinimide,phthalimide or maleimide; or

(iv) R₃, R₄, R₅, R₆, R₇, R_(A), R_(B), Q and n are as defined in any of(i)-(iii) or (v)-(viii),

V is as defined in (ii);

R₈ is H; and

R₁ and R₂ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisowhen more than one heteroatom is present, there is at least one carbonatom between each heteroatom, and

(b) R₁ and R₂ are unsubstituted or substituted with Y, preferably withV, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(v) R₁, R₂, R₅, R₆, R₇, R₈, Q and n are as defined in any of (i)-(iv) or(vi)-(viii);

V is as defined in (ii);

R₃ and R₄ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisowhen more than one heteroatom is present, there is at least one carbonatom between each heteroatom, and

(b) is unsubstituted or substituted with Y, preferably with V, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl;

(vi) R₁, R₂, R₃, R₄, R₇, R₈, Q and n are as defined in any of (i), (iv)or (v);

V is as defined in (ii);

R₅ and R₆ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisowhen more than one heteroatom is present, there is at least one carbonatom between each heteroatom, and

(b) R₅ and R₆ are unsubstituted or substituted with Y, preferably withV, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(vii) R₁, R₂, R₃, R₄, R₆ and R₈ are selected as in (i), (iv) or (v);

V is as defined in (ii);

n is zero; and

R₅ and R₇ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisowhen more than one heteroatom is present, there is at least one carbonatom between each heteroatom, and

(b) R5 and R₇ are unsubstituted or substituted with Y, preferably withV, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(viii) R₁, R₂, R₃, R₄, R₅, R₈ and Y are selected as in (i), (iv) or (v);

V is as defined in (ii);

R₆ and R₇, which are defined as in (ii), together with the atoms towhich each is attached form a bicyclic heterocycle or cyclic moiety,preferably, when n is zero, a reduced isoquinoline, and is preferably1,2,3,4,tetrahydroisoquinoline.

In all instances, unless specified, the carbon chains, which may bestraight or branched or cyclic, contain from 1 to about 1 2 carbonspreferably 1 to 6, and most preferably 4-6 carbons, and the cyclicmoieties preferably contain one ring or two fused rings with from 3 to16 atoms, preferably 4 to 12, with 4 to 6 in each ring, in the ringstructures.

The compounds of formulae (I), (II) and (III) are particularly usefulfor the treatment of neurodegenerative disorders and other disordersinvolving the accumulation of amyloid plaques.

Unless otherwise stated, the α-amino acids or analogs thereof of thecompounds of formulae I-III are preferably in their L-configuration. Intheir preferred configuration with reference to a particular compound,R₁, is S, R₃ is S, R₅ is R/S, and the chiral centers of X are R/S, R, Sor any combination thereof, and are preferably S.

Also, a compound of these formulae may be in free form, e.g., anamphoteric form, or a salt form, e.g., an acid addition or an anionicsalt. A compound may be converted to its salt or base form in anart-known manner, one from another. Prepared salts are trifluoracetate,hydrochloride, sodium, potassium or ammonium salts, although the scopeof the salts embraced is not limited thereto, the scope being extendedto include all of those salts known to be used in the art of chemistry.

Compounds are also provided herein. These compounds may be used in themethods. In certain embodiments, the compounds have formulae (I) or(II), as defined above, but with the proviso that: (1) at least one ofthe amino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid and X isa tertiary or secondary haloalkyl alcohol, R₁ is not the side chain ofcyclohexylalanine or cyclohexylglycine.

In other embodiments, when the compounds have formula (III), as definedabove, when X is a tertiary or secondary haloalkyl alcohol, then R₁ isthe side chain of a non-naturally-occurring α-amino acid and it is notthe side chain of cyclohexylalanine or cyclohexylglycine.

In certain other embodiments, the compounds have formulae (I) or (II),as defined above, but with the proviso that: (1) at least one of theamino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid, R₁ isnot the side chain of cyclohexylalanine or cyclohexylglycine.

In certain other embodiments, the compounds have formulae (I), (II) or(III) as defined above, but with the proviso that, when the compoundshave formula (I) or (II): R₁ is a subunit of a non-naturally-occurringamino acid, the side chain of R₁ is of a non-naturally-occurring aminoacid other than cyclohexylalanine or cyclohexylglycine, unless thecompounds are primary alcohols, then the non-naturally-occurring aminoacid is other than norleucine or norvaline.

Thus, in certain other embodiments in which the compounds are primaryalcohols, the compounds have formulae (I) or (II), particularly formulaI, as defined above, but with the proviso that: (1) at least one of theamino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid, it isnot the side chain of norleucine or norvaline.

Thus, in certain other embodiments in which the compounds are primaryalcohols, the compounds have formulae (I) or (II), particularly formulaI, as defined above, but with the proviso that: (1) at least one of theamino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid, it isnot the side chain of cyclohexylalanine, cyclohexylglycine, norleucineor norvaline.

In other embodiments, the compounds have formulae (I), (II) or (III) asdefined above, but with the proviso that, when the compounds haveformula (I) or (II): R₁ is a side chain of a non-naturally-occurringamino acid other than cyclohexylalanine or cyclohexylglycine, norleucineor norvaline.

In other embodiments, the compounds have formulae (I), (II) or (III) asdefined above, but with the proviso that, when the compounds haveformula (I) or (II): R₁ is a side chain of a non-naturally-occurringamino acid other than cyclohexylalanine or cyclohexylglycine,norleucine, norvaline, citrulline, ornithine, 4-phenyl-2-aminobutyricacid, 1-naphthylalanine, 2-naphthylalanine, sarcosine,2-indolinecarboxylic acid, β-alanine, β-valine, N-6-acetyllysine,O-4'-methyltyrosine, a substituted alanine and guanidinophenylalanine.

In certain other embodiments, the compounds have formulae (I), (II) or(III) as defined above, but with the proviso that, when the compoundshave formula (I) or (II): at least one of the amino acid residues in theresulting di-peptide or tri-peptide is a non-naturally-occurring α-aminoacid or at least one of the R₁, R₃ and R₅, preferably R₁, is a sidechain of a non-naturally-occurring amino acid, R₁ is notcyclohexylalanine, and at least one non-naturally occurring amino acid(or side chain thereof) is other than norleucine or norvaline, unlessthe resulting residue is a halo-substituted alcohol, particularlyfluoro-substituted alcohols. Such compounds include, but are not limitedto: (2SR)-(3SR)-N-Ac-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide and N-valeroyl-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide.

In other embodiments, the compounds have formulae (I) or (II) and atleast one of R₁, R₃ and R₅, preferably R₁ or R₅, includes at least oneunsaturated bond. Thus at least one of R₁, R₃ and R₅ is a straight orbranched carbon chain containing at least one unsaturated bond,preferably a double bond, and 2 to 10, preferably 3 to 7, morepreferably 4 to 6, carbon atoms in the chain. Such side chains include,but are not limited to substituted and unsubstituted propenes, butenes,pentenes, such as, 2-methyl-propenyl and 2-butenyl, which are among thepreferred residues.

The compounds provided herein are preferred for use in the methods,particularly the methods of treatment of cognitive disorders.

The compounds herein may be used in methods of identifying andclassifying proteolytic enzymes.

Pharmaceutical compositions containing a compound of formulae (II), (II)or (III) are provided. In particular, pharmaceutical compositionsformulated for single dosage administration are provided.

Combinations of compositions are also provided. The combinationscontain: (A) a composition containing one or more compounds of formula(I), (II) or (III) set forth above; and (B) a composition containingcompounds of copending U.S. application Ser. No. 08/369,422,particularly of formula (IV) or (V): ##STR4## or the hydrates andisosteres, diastereomeric isomers and mixtures thereof, orpharmaceutically acceptable salts thereof, wherein:

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(A), R_(B), Q and n are selected fromamong (i) , (ii), (iii), (iv), (v) , (vi), (vii) or (viii) as describedabove, X for the compounds of (B) is an aldehyde or ketone or preferablyselected from among --(CH₂)_(r) C(O)H, --(CH₂)_(r) C(O)haloalkyl,--(CH₂)_(r) C(O)(CH₂)_(r) CHN₂, --(CH₂)_(r) C.tbd.N, --C(CH₂)_(r)(O)C(CH₂)_(r) (O)OR_(D), --(CH₂)_(r) C(O)(CH₂)_(r) C(O)NR_(D) R_(D),(CH₂)_(r) C(OH)(CH₂)_(r) C(O)U, --(CH₂)_(r) C(OH)CH₂ C(O)U, --(CH₂)_(r)C(O)W and --(CH₂)_(r) C(O)CH₂ W; and U, W, and R_(D) are as describedabove.

Methods of inhibiting proteases, particularly serine, cysteine, andaspartyl proteases, particularly intracellular proteases are provided.In particular, the proteases are responsible for cleavage of APP toproduce Aβ.

Methods of treatment of diseases in which proteases play a pathologicalrole are provided. Among these diseases are cognitive disorders,hypertension, inflammatory disorders and others. The methods areeffected by administering an effective amount of the pharmaceuticalcompositions.

In particular, methods of treating a patient suffering from aneurodegenerative disease selected from among Alzheimer's disease,cognition deficits, Down's Syndrome, Parkinson's disease, cerebralhemorrhage with amyloidosis, dementia pugilistica, head trauma and anydisorder characterized by an accumulation of plaques in the brain, byadministering to the patient a therapeutically effective amount of acompound of formulae (I), (II) and (III) or compounds of formulae (I),(II) and (III) in which R₁, R₃, R₅ and R_(B) can all be side chains ofnaturally-occurring amino acids are provided.

Methods of treating a patient suffering from a disease statecharacterized by the degeneration of the cytoskeleton arising from athrombolytic or hemorrhagic stroke by administering a therapeuticallyeffective amount of a compound provided herein are also provided.

Methods of inhibiting proteases and methods of treatment of disorders,particularly neurodegenerative disorders using the combinations in which(A) and (B) are used simultaneously, successively or intermittently arealso provided.

Methods of identifying and classifying proteolytic enzymes are alsoprovided. These methods are effected by measuring the activity of anenzyme in the presence and absence of a compound provided andascertaining whether the enzyme activity is altered.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

As used herein, the term "alkyl" includes the straight, branched-chainand cyclic manifestations thereof, the number of carbons atoms isgenerally specified. Where not specified the alkyl groups preferablycontain from about 1 up to about 10 or 12, more preferably 1 to 6 or 7,and most preferably 4 to 6 carbons. Exemplary of such moieties aremethyl, ethyl, propyl, cyclopropyl, isopropyl, n-butyl, t-butyl,sec-butyl, cyclobutyl, pentyl, cyclopentyl, n-hexyl, n-nonal, n-decyl,cyclohexyl, cyclohexylmethyl, cyclohexylethyl, and the like. Lower alkylrefers to alkyl groups containing six or fewer carbon atoms.

As used herein, heteroatoms are selected from O, N or S.

The term "aryl"60 within the definitions of X, R_(B), R₁, R₃, R₅, and R₇includes carbocyclic and heterocyclic moieties. Preferred aralkyl andaryl moieties are phenyl, benzyl, phenethyl, 1- and 2-naphthylmethyl, 1-and 2-naphthyl, 2-, 3-, 4- pyridyl, 2- and 3-furyl, 1- and 2-indenyl, 1-and 2-thiophenyl, imidazolyl, indolyl, 2- and 3-thienyl, indole-3-ethyland the residue of 1,2,3,4,tetrahydroisoquinoline. Other carbocycles aresuch fused moieties as pentalenyl, indenyl, naphthaleneyl,naphthylmethyl, azulenyl, heptalenyl, acenaphthylenyl, 9-fluorenyl,phenalenyl, phenanthrenyl, anthracenyl, triphenylenyl, pyrenyl,chryrsenyl, and naphthacenyl. Exemplary of alkynyl is propynyl.Exemplary of alkenyl moieties are 2-methyl-2-propenyl,2-methyl-1-propenyl, propenyl, 1-butenyl, 2-butenyl, 3-butenyl,2,2-difluoroethenyl, as well as those straight and branched chainedmoieties having up to two double bonds. Cyclic carbon moietiespreferably contain one or two fused rings typically from 3 up to about16, preferably 4 up to about 12 carbons.

Haloalkyl embraces such moieties as --CF₃, --CF₂ CF₃, --CF₂ H, --CFH₂,CH₂ Cl and CH₂ Br and other halo substituted lower alkyls. Exemplary ofaryloxyalkenyl and aryloxyalkynyl moieties of R_(A) are phenoxymethyl,CF₃ -- substituted phenoxymethyl, benzyloxymethyl, phenoxybutyl-2-ene,1-phenyl-1-propene, CF₃ -phenoxybutyr-2-ene, and CF₃ -benzyloxymethyl.These moieties are preferred when R_(A) is other than R₁.

In those instances in which a substituent, such as the R₁, R₃, and/or R₅moiety, embrace the residue--or side chain--of a naturally occurringα-amino acid, it is to be noted that each α-amino acid has acharacteristic "R-group," the R-group being the residue--or sidechain--attached to the α-carbon atom of the amino acid. For example, theresidue of glycine is H, for alanine it is methyl, for valine it isisopropyl. The specific residues of the naturally occurring α-aminoacids are well known to those of skill in this art [see, e.g., A. L.Lehninger, Biochemistry: The Molecular Basis of Cell Structure andFunction, 1970 (or any edition thereafter), Worth Publishers, NY, see,particularly Chapter 4).

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, the moieties in the peptide analogs provided herein aredesignated according to the system of nomenclature in which the bindingregion of a proteinase is considered as a series of subsites, S, alongthe surface of the enzyme [see, Schecter and Berger (Biochem. Biophys.Res. Comm., 27, 157-162 (1967)]. Each subsite binds an individualpeptide residue, P. This system of nomenclature was originally designedfor papain, but has been adapted to other proteases. Thus, forconvenience and in keeping with the customary peptide designations, themoiety bearing the R₁ side chain (or residue) is designated as the P₁moiety, the moiety bearing the R₃ side chain (or residue) is designatedas the P₂ moiety, and that bearing the R₅ moiety is designated as the P₃moiety.

The N-terminal capping moieties represented by the R₇ --(Q)_(n) -- and(R_(B))--CH(R_(A))--(Q)_(n) -- include those moieties that protectmolecules from degradation by aminopeptidases and include, but are notlimited to, such generic groupings as arylcarbonyl, alkylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, aralkylsulfonyl,alkylsulfonyl, arylsulfonyl, and other equivalently functioning groupsknown in the art.

As defined particularly for the capping groups herein, eitherindividually or as a part of a larger group, "alkyl" means a linear,cyclic, or branched-chain aliphatic moiety of 1 to 20 carbon atoms;"aryl" means an aromatic moiety, e.g., phenyl, of 6 to 18 carbon atoms,unsubstituted or substituted with one or more alkyl, substituted alkyl,nitro, alkoxy, or halo groups; "substituted alkyl" means an alkyl grouphaving a substituent containing a heteroatom or heteroatoms such as N,O, or S; "halo" means Cl or Br; and "alkaryl" means an aryl moiety of 6to 19 carbon atoms having an aliphatic substituent, and, optionally,other substituents such as one or more alkyl, substituted alkyl, alkoxyor amino groups.

Examples of suitable N-terminal blocking groups include, but are notlimited to, formyl, t-butyloxycarbonyl, isopropyloxycarbonyl,allyloxycarbonyl, acetyl, trifluoracetyl, methyl, ethyl, benzyl,benzoyl, acetoacetyl, chloroacetyl, succinyl, phthaloxy,phenoxycarbonyl, methoxysuccinyl, p-methoxybenzenesulfonyl,p-toluenesulfonyl, isovaleroyl, methanesulfonyl, benzyloxycarbonyl,substituted benzyloxycarbonyl, adipyl, suberyl, thalamido-, morpholino-,azelayl, dansyl, tosyl, 2,4-dinitrophenyl, fluorenylmethoxycarbonyl,methoxyazelayl, methoxyadipyl, methoxysuberyl, 1-adamantanesulfonyl,1-adamantaneacetyl, 2-carbobenzoyl, phenylacetyl, t-butylacetyl,bis[(1-methyl)methyl]acetyl, and thioproline.

As used herein, an effective amount of a compound for treating adisorder is an amount that is sufficient to ameliorate, or in somemanner reduce a symptom or stop or reverse progression of a condition.Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective.

As used herein, treatment means any manner in which the symptoms orpathology of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography [TLC], gelelectrophoresis and high performance liquid chromatography [HPLC], usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures.

As used herein, pharmaceutical activity refers to the activity of thecompounds herein to treat a disorder.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular compound that achieves a 50% inhibition of a maximalresponse.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, a prodrug is a compound that, upon In vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, once apharmaceutically active compound is identified, those of skill in thepharmaceutical art generally can design prodrugs of the compound [see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392].

As used herein, amyloid precursor protein (APP) is the progenitor ofdeposited amyloidogenic Aβ peptides (Aβ) found in the senile plaques inpatients with diseases, such as Alzheimer's disease (AD), that arecharacterized by such deposition. α-sAPP is an alternative cleavageproduct of APP; its formation precludes formation of Aβ.

As used herein, Cha is cyclohexylalanine, and Chg is cyclohexylglycine.

As used herein, the abbreviations for any substituent groups, protectivegroups, amino acids and other compounds, are, unless indicatedotherwise, in accord with their common usage, recognized abbreviations,or the IUPAC-IUB Commission on Biochemical Nomenclature [see, (1972)Biochem. 11:1726]. Some exemplary abbreviations include: BOC ist-butyloxycarbonyl; BOP is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; DCC is dicyclohexylcarbodiimide; DDZ isdimethoxydimethylbenzyloxy; DMT is dimethoxytrityly; FMOC isfluorenylmethyloxycarbonyl; and TFA is trifluoroacetic acid.

A. The tri- and dipeptide analogs and amino acid analogs

Compounds of formulae (I), (II) and (III): ##STR5## or the hydrates andisosteres, diastereomeric isomers and mixtures thereof, orpharmaceutically acceptable salts thereof, in which X is selected asdescribed above; and R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(A), R_(B) Q andn are selected from among (i), (ii), (iii), (iv), (v), (vi), (vii) or(viii) as described above, but with the proviso that, when the compoundshave formula (I) or (II): (1) at least one of the amino acid residues inthe resulting di or tri-peptide is a non-naturally-occurring α-aminoacid or at least one of the R₁, R₃ and R₅ is not a side chain of anaturally-occurring amino acid; and (2) when R₁ is the side chain from anon-naturally occurring amino acid and X is a tertiary or secondaryhaloalkyl alcohol, R₁ is not the side chain of cyclohexylalanine orcyclohexylglycine.

In other embodiments, when the compounds have formula (III), as definedabove, when X is a tertiary or secondary haloalkyl alcohol, then R₁ isthe side chain of a non-naturally-occurring α-amino acid and it is notthe side chain of cyclohexylalanine or cyclohexylglycine.

In certain other embodiments, the compounds have formulae (I) or (II),as defined above, but with the proviso that: (1) at least one of theamino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid, R₁ isnot the side chain of cyclohexylalanine or cyclohexylglycine.

In certain other embodiments, the compounds have formulae (I), (II) or(III) as defined above, but with the proviso that, when the compoundshave formula (I) or (II): R₁ is a subunit of a non-naturally-occurringamino acid, the side chain of R₁ is of a non-naturally-occurring aminoacid other than cyclohexylalanine or cyclohexylglycine, unless thecompounds are primary alcohols, then the non-naturally-occurring aminoacid is other than norleucine or norvaline.

Thus, in certain other embodiments in which the compounds are primaryalcohols, the compounds have formulae (I) or (II), particularly formulaI, as defined above, but with the proviso that: (1) at least one of theamino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid, it isnot the side chain of norleucine or norvaline.

Thus, in certain other embodiments in which the compounds are primaryalcohols, the compounds have formulae (I) or (II), particularly formulaI, as defined above, but with the proviso that: (1) at least one of theamino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid, it isnot the side chain of cyclohexylalanine, cyclohexylglycine, norleucineor norvaline.

In other embodiments, the compounds have formulae (I), (II) or (III) asdefined above, but with the proviso that, when the compounds haveformula (I) or (II): R₁ is a side chain of a non-naturally-occurringamino acid other than cyclohexylalanine or cyclohexylglycine, norleucineor norvaline.

In other embodiments, the compounds have formulae (I), (II) or (III) asdefined above, but with the proviso that, when the compounds haveformula (I) or (II): R₁ is a side chain of a non-naturally-occurringamino acid other than cyclohexylalanine or cyclohexylglycine,norleucine, norvaline, citrulline, ornithine, 4-phenyl-2-aminobutyricacid, 1-naphthylalanine, 2-naphthylalanine, sarcosine,2-indolinecarboxylic acid, β-alanine, β-valine, N-6-acetyllysine,O-4'-methyltyrosine, a substituted alanine and guanidino-phenylalanine.

In certain other embodiments, the compounds have formulae (I), (II) or(III) as defined above, but with the proviso that, when the compoundshave formula (I) or (II): at least one of the amino acid residues in theresulting di-peptide or tri-peptide is a non-naturally-occurring α-aminoacid or at least one of the R₁, R₃ and R₅, preferably R₁, is a sidechain of a non-naturally-occurring amino acid, R₁ is notcyclohexylalanine, and the at least one non-naturally occurring aminoacid (or side chain thereof) is other than norleucine or norvaline,unless the resulting residue is a halo-substituted alcohol, particularlyfluoro-substituted alcohols. Such compounds include, but are not limitedto: (2SR)-(3SR)-N-Ac-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide and N-valeroyl-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide.

In other embodiments, the compounds have formulae (I) or (II) and atleast one of R₁, R₃ and R₅, preferably R₁ or R₅, includes at least oneunsaturated bond. Thus at least one of R₁, R₃ and R₅ is a straight orbranched carbon chain containing at least one unsaturated bond,preferably a double bond, and 2 to 10, preferably 3 to 7, morepreferably 4 to 6, carbon atoms in the chain. Such side chains include,but are not limited to substituted and unsubstituted propenes, butenes,pentenes, such as, 2-methyl-propenyl and 2-butenyl, which among thepreferred residues.

Preferred among these compounds, subject to or defined with any of theprovisos, are those in which:

R₁ is preferably H or a straight or branched chain carbon chaincontaining 2 to 6 carbons and one unsaturated, preferably a double bond,or is a cyclic moiety containing from 5 to 6 members, and is morepreferably methyl, 2-methyl propene, 2-butene, cyclohexyl, loweralkyl-substituted cyclohexyl or cyclohexylmethyl, hydroxyphenyl,isopropyl, toluyl, t-butyl, isobutyl, n-butyl, 1-aminobutyl,methylethylthioether and is more preferably n-butyl, toluyl, isobutyl orcyclohexylmethyl;

R₂, R₄ and R₈ are each independently selected from among H or C₁₋₄alkyl, and more preferably methyl or ethyl;

R₃ is H, C₁₋₄ alkyl, aryl, particularly, phenyl, naphthyl andhydroxyphenyl, 1-aminobutyl, acetamide, and more preferably iso-butyl orphenyl or toluyl;

R₅ is C₁₋₄ alkyl, and more preferably iso-butyl;

R₆ is H or C₁₋₄ alkyl, and more preferably H or methyl;

R₇ --(Q)_(n) is acyl, benzyloxycarbonyl (Cbz),9-fluorenylmethylcarbonate (Fmoc), Ac, BOC, tosyl, with Cbz, Ac and Fmocbeing more preferred, and Cbz and Ac most preferred;

Q is --C(O)--, --S(O)₂ -- and --O--C(O), with --C(O)-- and --O--C(O)being more preferred, and --O--C(O) most preferred;

R₈ is C₁₋₄ alkyl or C₂₋₄ alkenyl, and more preferably iso-butyl;

R_(A) is --(T)_(m) --(D)_(m) --R₁, in which T is oxygen, carbon, ornitrogen, with oxygen or carbon being more preferred, and D is C₁₋₄alkyl or C₂₋₄ alkenyl, with a mono-unsaturated C₃₋₄ alkenyl being morepreferred; and

X, which is as defined above, is preferably a secondary alcohol, andmore preferably least one of A or B is H and the other is haloalkyl, inwhich the carbon chain is straight branched or cyclic, and is preferablya lower alkyl containing 1-6 carbons, such as CF₃, C₂ F₅.

Also among preferred compounds are those of formulae (I), (II) and (III)in which R_(B) and R_(A) and the atom to which each is attached form(2SR)-N-[(2S)-2-benzoxy-4-methylpentanoyl] or(2SR)-N-[(2R)-[2-(1'-phenyl-1'-propene)-4-methylpentanoyl or valeroyl.

When R₁, R₃, and R₅ are a side-chain from other than a residue of anaturally occurring α-amino acid, it is preferred that such moiety is astraight or branched carbon chain, preferably containing at least oneunsaturated bond, preferably a double bond, and 2 to 10, preferably 4 to7, more preferably 4-6 carbon atoms in the chain, such as, but notlimited to, 2-methyl propene and 2-butene, or is a cylic moiety,preferably containing 4-6 members, more preferably is cyclohexyl orcyclohexylmethyl. The resulting residues including such moietiesinclude, but are not limited to, 2-amino-4-methyl-4-pentenoic acid,2-amino-4-hexenoic acid, cyclohexylalanine and cyclohexylglycine,(2S)-2-amino-4-methyl-4-pentenoic acid and (2S)-2-amino-4-hexenoic acid.

When the compounds are used in the methods of treating neurodegenerativediseases and cognitive disorder provided herein, the side chains fromnorvaline and leucine or from norvaline and norleucine are alsopreferred.

In particular, preferred compounds are those in which at least one ofR₁, R₃, and R₅ is 2-methyl-propene, 2-butene, cyclohexyl orcyclohexylmethyl. More preferred are those in which R₁, R₃, and R₅ are2-methyl-propene, 2-butene, cyclohexyl or cyclohexylmethyl, and X is--CH(OH)C_(k) H.sub.(2k+1-m) F_(m) C in which k is 1-6, preferably 1-3,m is 0 to 2k+1; --CH(OH)C₆ H.sub.(5-q))F_(q) in which q is 0 to 5,--(CH₂)_(r) C(OH)CF₃, CH(OH)CF₃, CH(OH)CHN₂ and CH(OH)CF₃.

Preferred heterocyclic ring moieties containing R₁ and R₂ and the atomsto which they are attached, when R₈ is H, are morpholino,thiomorpholino, pyrrolidinyl, or V-substituted pyrrolidinyl,particularly 4-hydroxy pyrrolidinyl.

Preferred heterocyclic ring moieties containing R₆ and R₇ and the atomsto which they are attached when (Q)n is a carbonyl group are selectedfrom among succinimide, phthalimide or maleimide, with phthalamide beingmore preferred.

Preferred heterocyclic ring moieties containing R₆ and R₇ and the atomsto which they are attached when n in (Q)_(n) is zero are morpholino,thiomorpholino, pyrrolidinyl, V-substituted pyrrolidinyl, particularly4-hydroxy pyrrolidinyl, or 1,2,3,4-tetrahydroisoquinoline.

Preferred moieties, when n is zero, and when R₃ and R₄ or R₅ and R₇ andtaken together with the atoms to which they are attached formheterocyclic moieties are morpholino, thiomorpholino, pyrrolidinyl, orV-substituted pyrrolidinyl, particularly 4-hydroxy pyrrolidinyl.

The following are among the preferred compounds provided herein:(2SR)-N-Cbz-L-Leu-L-Leu N-[2-(4-methyl-4-pentenol)]amide,(1SR)-(2S)-N-Cbz-L-Leu-L-Leu N-[2-(thiazole-hexanol)]amide,(2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol,(2SR)-(3SR)-N-Ac-L-Leu-L-Leu N-[3-(1,1,1-trifluoro-2-heptanol)]amide,(2SR)-(3SR)-N-Cbz-L-Leu N-[3-(1,1,1-trifluoro-2-heptanol)]amide,(2SR)-(3SR)-N-valeroyl-L-Leu N-[3-(1,1,1-trifluoro-2-heptanol)]amide,(SR)-N-Cbz-L-Leu-L-Leu N-[2-(phenylhexanol)]amide,(2SR)-(3S)-N-Cbz-L-Leu N-[3-(1,1,1-trifluoro-2-butanol)]amide,(3SR)-(4S)-N-Cbz-L-Leu-L-Leu N-[4-(ethyl2,2-difluoro-3-hydroxyoctanoate)]amide, (4S)-(3SR)-N-Cbz-L-Leu-L-LeuN-[4-(1,1,1-trifluoro-2,2-difluoro-3-octanol)]amide,(3SR)-(4S)-N-Cbz-L-Leu-L-LeuN-[4-(1,1,1-trifluoro-2,2-difluoro-3-methyl-3-octanol)]amide,(2SR)-(3SR)-N-Cbz-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-methyl-2-heptanol)]amide,(2SR)-(3S)-N-Cbz-L-Leu-L-LeuN-[3-(1-(1'-phenyl-3'(trifluoromethyl)-pyrazoloxy)-2-heptanol)]amide,(2SR)-H-L-Leu N-[2-(ethyl 4-methyl-4-pentenoate)]amide hydrochloride,(2SR)-N-[(2S)-2-benzoxy-4-methylpentanoyl]-L-LeuN-[2-(4-methyl-4-pentenol)]amide, (2SR)-(3S)-N-Cbz-L-Leu-LeuN-[3-(2-hydroxy-heptanoic acid)]amide, (2SR)-(3S)-N-Cbz-L-Leu-L-LeuN-[3-(methyl 2-hydroxy-heptanoate)]amide, (2SR)-(3S)-N-Cbz-L-Leu-L-LeuN-[3-(benzyl 2-hydroxy-heptamide)]amide, (3SR)-(4S)-N-Cbz-L-Leu-L-LeuN-[4-(benzyl 3-hydroxy-octamide)]amide, (2SR)-(3S)-N-Cbz-L-Leu-L-LeuN-[3-(1-furfylthio-2-heptanol)]amide,(2SR)-N-[(2R)-[2-(1'-phenyl-1'-propene)-4-methylpentanoyl]]-L-Leu-N-[2-(4-methyl-4-pentenol)]amide,(2SR)-N-Ac-L-Leu-L-Leu-N[2-(trans-4-hexanol)]amide,(2SR)-N-Ac-L-Leu-L-Leu N-[2-(4-methyl-4-pentenol)]amide,(2SR)-N-Ac-L-Leu-L-Leu N-[2-(4-methyl-4-pentenol)1]amide,N-dansyl-L-Leu-L-Leu-DL-norleucinol, andN-Ac-L-Phe-L-Leu-DL-norleucinol.

Particularly preferred compounds include: (2SR)-(3SR)-N-Ac-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide and (2SR)-(3SR)-N-valeroyl-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide.

B. Synthesis of the tri- and dipeptide analogs and amino acid analogs

1. Reaction schemes

The following reaction schemes are depicted to illustrate theconstruction of the peptides provided herein and to illustrate thevariety of reactions that may be used to prepare the intermediates fromwhich compounds of formulae (I), (II) and (III) may be prepared.##STR6##

Reaction Scheme A illustrates the preparation of compounds of formulae(I), (II) and (III) in which the X moiety is a primary alcohol. Ineffecting the preparations, a standard Fischer esterification of theappropriate amino acid precursors produces the analogous esters (2) thatare coupled with the appropriate N-protected P₂ P₃ moieties (3)utilizing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC; as its HClsalt) in the presence of hydroxybenzyl triazole hydrate (HOBt) andtriethylamine (Et₃ N). The reaction is conducted in CH₂ Cl₂ at roomtemperature under argon or nitrogen under anhydrous conditions. Theresulting compounds (4) are reduced to the desired alcohols usingstandard reduction conditions, including, but not limited to, the use oflithium boro-hydride (LiBH₄) in tetrahydrofuran (THF) at about 0° C. inan inert atmosphere (Ar or N₂) under anhydrous conditions. Analogousdipeptides and N-protected amino acid derivatives are prepared, usingsubstantially the same procedure, but replacing the P₂ P₃ moieties[compound 3, Reaction Scheme A, hereinafter A-3] with the appropriate P₂moiety or N-capping group. As noted above, the moiety bearing the R₁side chain (or residue) is designated as the P₁ moiety, the moietybearing the R₃ side chain (or residue) is designated as the P₂ moiety,and that bearing the R₅ moiety is designated as the P₃ moiety.

Reaction Scheme B depicts an exemplary method for the preparation ofprecursors of reactants, for the preparation of compounds for use in themethods herein, that are amenable to substitutions on the α-carbon atom.The substituents, include, but are not limited to residues of naturally-and non-naturally-occurring α-amino acids. In this scheme,N-(diphenylmethylene)glycine ethyl ester (1) is treated with lithiumbis(trimethylsilyl)amide in THF under an inert atmosphere attemperatures of about -78° C., and the in situ generated base is reactedwith the appropriate alkyl halide to effect a nucleophilic displacement.The so-alkylated intermediates (2) and (4) are subjected to hydrolysisto produce the amines (3) and (5) that are available for appropriate usein the construction of the desired dipeptides and tripeptides in whichR₁, R₃ or R₅ are side chains other than those of naturally occurringα-amino acids. Similarly, this scheme may be used to prepare compoundsin which R₂, R₄ and/or R₆ are an alkylated product.

Alternatively, precursor reactants may be prepared followingsubstantially the same procedure described by O'Donnell et al.[O'Donnell et al. (1994) Tetrahed. Lttrs. 35:9383-9386].

Reaction Scheme C illustrates the preparation of the P₁ moiety in whichX is C(OH)W. In effecting this preparation, the N-methoxy-N-methylamidederivative (1) is reduced with lithium aluminum hydride under anhydrousconditions in an inert atmosphere at 0° C. to produce the correspondingaldehyde (2). The difluoro-hydroxy esters [compounds (2)] are producedby a standard Reformatsky reaction [see, e.g., Rathke (1975) Org. React.22:423-460; and March (1985) Advanced Organic Chemistry, 3d Ed., J.Wiley & Sons] followed by deprotection by hydrolysis with 4 N HCl indioxane to obtain compounds (3). Compounds (3) are coupled to theappropriate P₂ P₃ moieties (compound A-3) to obtain desired tripeptidederivatives (4). Compounds (3) may be coupled with appropriate P₂moieties, such as (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂ (R₃)C(O)OH), toobtain the desired dipeptides, or with appropriate N-capping moieties toobtain desired N-protected amino acid derivatives.

Reaction Scheme D illustrates the preparation of compounds having a P₁moiety in which X is C(O)W, as defined above, C₁₋₆ alkyl or aralkyl. Ineffecting the preparations, the N-methoxy-N-methylamide derivative (1)is treated with a lithiothiazole nucleophile generated in situ toproduce a ketothiazole, which is deprotected by hydrolysis with 4 N HClin dioxane to obtain compounds (2). Compounds (2) are then either a)coupled to appropriate P₂ P₃ moieties (A-3) to obtain desiredderivatives of compound (3); or b) are coupled to appropriate P₂moieties, such as, but not limited to, (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂(R₃)C(O)OH), to obtain the desired dipeptides; or c) are coupled toappropriate N-capping moieties to obtain desired N-protected amino acidderivatives. Subsequent reduction of the ketone using sodium borohydridein methanol affords the desired alcohol (4). The corresponding aminoacid analogs and di- and tri-peptide derivatives may be obtained byreplacing the lithio derivative of thiazole with litho derivatives ofother aryl, aralkyl and alkyl moieties and by following substantiallythe same procedures.

Reaction Scheme E illustrates the preparation of compounds of formulae(I), (II) and (III) in which the X moiety is a halomethyl alcohol. Thereaction is initiated by reacting an R₁ -substituted nitromethane with atrifluoromethyl acetal (2) in N,N-dimethylformamide (DMF) in thepresence of potassium carbonate at about 60° C. to yield a1,1,1-trifluoro-2-hydroxy-3-nitro derivative (3) which are reduced withH₂ in the presence of Raney Nickel to yield the corresponding amines(4). By appropriate coupling to the appropriate dipeptide (A-3), thedesired trifluoromethyl alcohols (6) of formula I may be produced. Thecorresponding dipeptide derivatives of the mono-, di- andtri-fluoromethyl alcohols of formulae (I)-(III) may be produced byselecting the appropriate N-capping group or N-protected amino acidderivative. Furthermore, by use of mono- and difluoromethyl analogs offormula (2) and by following substantially the same procedures,corresponding --CH₂ F and --CHF₂ alcohol analogs of formulae (I), (II)or (III) are produced.

Alternatively, compounds (C-1) can be reduced under standard anhydrousconditions, lithium aluminum hydride in THF at about 0° C. under inertatmosphere, affords the corresponding aldehyde. The aldehyde is treatedwith trifluoromethyltrimethylsilane under inert atmosphere in THF atabout 0° C. in the presence of tetrabutylammoniumfluoride [see,Krishnamurti et al. (1991) J. Org. Chem 56:984-989], and thendeprotected by hydrolysis with 4 N HCl in dioxane to obtain compounds(6). Compounds (6) are then either a) coupled to appropriate P₂ P₃moieties (A-3) to obtain desired tripeptide derivatives (6); or b) arecoupled to appropriate P₂ moieties, such as, but not limited to,(R_(A))CH(R_(B))--C(O)N(R₄)--CH₂ (R₃)C(O)OH), to obtain the desireddipeptides; or c) are coupled to appropriate N-capping moieties toobtain desired N-protected amino acid derivatives.

Reaction Scheme F illustrates the preparation of compounds of formulae(I), (II) and (III) in which the X moiety is pentafluoromethyl alcohol.To effect preparation of these compounds, the N-methoxy-N-methylamidederivatives (1) are treated with pentafluoroethyl lithium, generated insitu, to produce the pentafluoroethyl ketones (2), which are deprotectedby hydrolysis with 4 N HCl in dioxane to obtain compounds (3). Compounds(3) are either a) coupled to the appropriate P₂ P₃ moieties (A-3) toobtain desired tripeptide derivatives; or b) coupled to appropriate P₂moieties, such as, but not limited to, (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂(R₃)C(O)OH) to obtain the desired dipeptides; or c) coupled withappropriate N-capping moieties to obtain the desired N-capped amino acidderivatives. The resulting pentafluoromethyl derivatives are reducedwith sodium borohydride in methanol to afford the desired alcohols (4).

Reaction Scheme G illustrates the preparation of compounds of formulae(I), (II) and (III) in which the X moiety is a diazomethane that can beconverted to a haloalkyloyl or haloaryloyl alcohol. In this process, theamine-protected amino acid derivatives and peptides or peptide analogsare reacted with isobutyl chloroformate in the presence of4-methylmorpholine in CH₂ Cl₂ at -78° C. The anhydride derivatives arereacted with diazomethane according to standard procedures known in theart. If desired, the diazoketones may be treated with an appropriateacid (such as, but not limited to HBr), in pyridine to afford compounds(2), which are treated with the appropriate alkyl or aryl alcohol in DMFunder alkali conditions to produce the corresponding alkyloyl andaryloyl ketones. The resulting ketones are reduced with sodiumborohydride in methanol to produce the desired alcohols. The alcoholsare treated with H₂ /Pd(C) in methanol to afford (3). Compounds (3) arethen either a) coupled to appropriate P₂ P₃ moieties (A-3) to obtaindesired derivatives of compound (3); or b) are coupled to appropriate P₂moieties, such as, but not limited to, (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂(R₃)C(O)OH), to obtain the desired dipeptides; or c) are coupled toappropriate N-capping moieties to obtain desired N-protected amino acidderivatives.

Reaction Scheme H illustrates the preparation of compounds in which theX moiety is a fluoro-substituted aryl alcohol. In effecting this scheme,the N-methoxy-N-methylamide derivatives (1) are treated with theappropriate fluoro(1-5)-substituted bromobenzene moiety in N-butyllithium at -78° C. under anhydrous conditions in an inert atmosphere.This generates fluorobenzene lithium in situ, to produce afluoro-substituted aromatic ketones (2), which are deprotected byhydrolysis with 4 N HCl in dioxane to obtain compounds (3). By couplingcompounds (3) to the appropriate P₂ P₃ moieties (A-3), the correspondingfluoro-substituted aromatic ketone tripeptides (4) are produced. Theketone tripeptides are reduced with sodium borohydride in methanol toafford the desired corresponding alcohols (4). Analogous dipeptides andN-protected amino acid derivatives may be prepared using substantiallythe same procedure, but replacing the P₂ P₃ moieties with theappropriate P₂ moiety or N-capping group.

Reaction Scheme I illustrates the formation of a dipeptide in whichR_(B) is aryloxy, aralkoxy or an alkoxy in an enantiomeric pure isomer.The reaction is initiated by a two step process in which: (a) compounds(1) are deaminated by treatment with NaNO₂ in HCl; and (b) the resultingacids are esterified with an alkyl halide in the presence of DMF andcesium carbonate to produce compounds (2). These are treated with a2,2,2-trichloroacetimate derivatives in the presence oftrifluoromethanesulfonic acid in CH₂ Cl₂ to obtain the correspondingester. The ester moieties are hydrolyzed with lithium hydroxide inperoxide and a methanol-water solvent to produce the enantiomers (3).The isomers are coupled with the appropriate P₂ P₁ moieties [as esters;e.g., compounds (A-3)], and the resulting esters (5) are reduced to thedesired alcohols (6).

Reaction Scheme J illustrates the preparation of compounds of formulae(I), (II) and (III) in which X is C(O)CH₂ Y. The N-protected diazoketonederivatives of compound (1) are subjected to an addition reaction withan hydrohalic acid, preferably HCl, in pyridine to produce halomethylderivatives (2), which are subjected to a nucleophilic displacementreaction using an activated anion of the desired Y moiety, (e.g., Y), toafford compounds (3). Standard hydrogenation reactions remove theN-protecting group followed by the above-described coupling procedureswith the desired P₂ P₃ moieties (A-3) to produce compounds (5), whichare reduced to the desired alcohols (6). Analogous dipeptides andN-protected amino acid derivatives may be prepared using substantiallythe same procedure but replacing the P₂ P₃ moieties with the appropriateP₂ moiety or N-capping group.

Reaction Scheme K illustrates the preparation of compounds of formulae(I), (II) and (III) in which X is selected from moieties (a)--CH(OH)--C(O)--NR_(D) R_(D) and (b) CH(OH)--C(O)OR_(D). The processstarts by obtaining aldehyde (2) by reducing the N-methoxy-N-methylamidederivative (1), followed by preparation of the cyanohydrin (3), which ishydrolyzed to its free acid (4) using standard and known reactiontechniques. Coupling of the appropriate P₂ P₃ moiety (A-3) to the acid(4) is effected by the use of an activated isobutyl chloroformate in thepresence of 4-methylmorpholine at -78° C. in an inert atmosphere underanhydrous conditions to afford the acid (5). The acid (5) may beesterified to its corresponding ester or may be coupled with an amine(NR_(D) R_(D)) to produce the desired amide (6). Analogous dipeptidesand N-protected amino acid derivatives may be prepared usingsubstantially the same procedure, but replacing the P₂ P₃ moieties withthe appropriate P₂ moieties or N-capping groups.

Alternatively, compounds (2) may be transformed to their N-protected(preferably protected by a BOC group) alkyl ester by reaction withethylacetate in the presence of LDA to produce compounds (8) which arehydrolyzed with 4 N HCl in dioxane to remove the protecting group toproduce the corresponding β-hydroxy ethyl esters (9). These esters arethen coupled with compounds (A-3) and the resulting compounds arehydrolyzed to their β-hydroxy acids or they may be coupled to form theirβ-hydroxyamides of compounds (11).

Reaction Scheme L illustrates the process by which compounds of formulae(I), (II) and (III) in which R₂, R₄, or R₆ are an R_(D) moiety otherthan H. This procedure uses standard N-protection,N-alkylation-esterification and de-protection procedures such as thoseexemplified in the depicted schemes. Although the reaction schemedepicts N-alkylation at the projected P₁ moiety, any of the P₂ and P₃moieties may be similarly N-alkylated by appropriate selection of thestarting materials followed by the coupling procedures required toconstruct the desired peptides and amino acid derivatives of formulae(I), (II) and (III).

Reaction Scheme M illustrates the process by which compounds of formulae(I), (II) and (III) in which X is a tertiary alcohol, particularlyhaloalkyl-substituted tertiary alcohols are prepared. Compounds (F-2)[see, Scheme F] are converted to tertiary alcohols, such as apentafluoroethyl-substituted tertiary alcohol, by treatment with ananalogous Grignard or lithium derivative by standard procedures known inthe art. These compounds are deprotected with 4 N HCl in dioxane toproduce compounds (2). Compounds (2) are either a) coupled to theappropriate P₂ P₃ moieties (A-3) to obtain desired tripeptidederivatives; or b) coupled to appropriate P₂ moieties, such as, but notlimited to, (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂ (R₃)C(O)OH) to obtain thedesired dipeptides; or c) coupled with appropriate N-capping moieties toobtain the desired N-capped amino acid derivatives. Using analogousmethods, but starting with the trifluoromethyl derivative of (F-2) orother such derivative, the trifluoromethyl or corresponding derivativemay be prepared.

Reaction Scheme N illustrates another process by which compounds offormulae (I), (II) and (III) in which X is a tertiary alcohol areprepared. The precursors (E-6) are treated with (BOC)₂ under standardamine-protecting conditions, followed by oxidation with the Dess-Martinreagent of the trifluoromethyl alcohol to give N-BOC trifluoromethylketones (1). Reaction of (1) with the appropriate Grignard or lithiumderivative affords the tertiary alcohol (2). Deprotection by hydrolysiswith 4 N HCl in dioxane produces (3). Compounds (3) are either a)coupled to the appropriate P₂ P₃ moieties (A-3) to obtain desiredtripeptide derivatives (4); or b) coupled to appropriate P₂ moieties,such as, but not limited to, (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂(R₃)C(O)OH) to obtain the desired dipeptides; or c) coupled withappropriate N-capping moieties to obtain the desired N-capped amino acidderivatives.

Throughout the above presentation of the methods useful for preparingthe compounds herein, particularly as it relates to the foregoingreaction schemes, the full embodiment of the entire scope of thecompounds (as defined in formulae (I), (II) and (III)) was not depictedwithin all of the structures illustrated for each of the reactants andend-products. The state of the art is such that one of skill in the artwould be able to extend these specific illustrations to embrace theimplied generic teachings by the use of analogy reasoning to prepare thedesired compounds embraced within the scope of formulae (I), (II) and(III). For example, one of skill in the art could utilize the finalproduct of Reaction Scheme A in preparing any of the compounds offormulae (I), (II) and (III) bearing the R₁ side chain functionality,which is other than a residue of a naturally occurring α-amino acid.Similarly, in Reaction Scheme D, the preparation of a thiazolederivative is achieved by coupling the N-methoxy-N-methylamidederivative of a precursor for preparing a depicted tripeptide (3). Adipeptide bearing a thiazole derivative could be prepared by theapplication of the analogy reasoning possessed by a person of skill inthe art. Similarly, lithio derivative of another heterocycle in which Xis C(O)aryl and the aryl moiety is other than a thiazole embraced withinthe scope of the compounds herein could be prepared.

Thus, the scope of those compounds that can be prepared by the methodsof the foregoing reaction schemes is not limited to the specificcompounds depicted but rather to those compounds defined by formulae(I), (II) and (III) using the teachings already available in the art andin the disclosure herein, including the above-discussion and Examplesbelow.

2. Procedures to effect the reaction schemes

The construction of the tri- and dipeptide analogs and amino acidanalogs of Formulae (I), (II) and (III) may be effected using proceduresand techniques known in the art and described herein. Many of thenecessary starting materials and the reactants utilized are known andmay also be commercially available. In those instances in which they arenot generally available, they may readily be generated by analogous useof known chemical processes and techniques readily available in thescientific and patent literature or as described herein.

As the reaction schemes depicted herein (Schemes A-N) extensivelyutilize coupling and oxidation procedures, the following elaborates avariety of the procedures that may be functional alternatives to thosespecifically mentioned within the depicted schemes. As a preferredoxidation procedure, the Swern oxidation is effected by reacting 2 to 10equivalents of dimethyl sulfoxide (DMSO) with about 1 to 6 equivalentsof trifluoroacetic anhydride [(CF₃ CO)₂)] or oxalyl chloride [--(COCl)₂]. The reactants are dissolved in an inert solvent, e.g., methylenechloride (CH₂ Cl₂), the reactor is under an inert atmosphere underanhydrous conditions at temperatures of about -80° C. to -50° C. to forman in situ sulfonium adduct to which is added about 1 equivalent of thealcohols (e.g., A-4). Preferably, the alcohols are dissolved in an inertsolvent, e.g., CH₂ Cl₂ or minimum amounts of DMSO, and the reactionmixture is allowed to warm to about -50° C. (for about 10-20 minutes)and then the reaction is completed by adding 3 to 10 equivalents of atertiary amine, e.g., triethylamine, N-methyl morpholine, etc. Followingoxidation, the desired intermediates are isolated and are ready for thenext step in the reaction sequence.

A modified Jones oxidation procedure may conveniently be effected byreacting the alcohols with pyridinium dichromate by contacting thereactants in a water-trapping sieve powder, (e.g., a grounded 3 Angstrommolecular sieve) in the presence of glacial acetic acid at about 5° C.to 50° C., preferably at room temperature.

Alternatively, 1 to 5 equivalents of a chromic anhydride-pyridinecomplex [i.e., a Sarett reagent prepared in situ (see, e.g., Fieser andFieser "Reagents for Organic Synthesis" Vol. 1, pp. 145 and Sarett, etal., J.A.C.S. 25, 422 (1953))] that is prepared in situ in an inertsolvent (e.g., CH₂ Cl₂) in an inert atmosphere under anhydrousconditions at about 0° C. to 50° C. to which complex is added 1equivalent of the alcohols allowing the reactants to interact for about1 to 15 hours, followed by the isolation of the desired product.

Another alternative process for the converting of alcohols to thedesired ketones is an oxidation reaction that employs periodane [i.e.,1,1,1-triacetoxy-1,1-dihydro, 1,2-benzoxidol 3-(1-H)-one (see DessMartin, J. Org. Chem. 48, 4155, (1983))]. This oxidation is effected bycontacting 1 equivalent of the alcohols with 1 to 5 equivalents ofperiodane (preferably 1.5 equivalents) in suspension in an inert solvent(such as, but not limited to CH₂ Cl₂) under an inert atmosphere(preferably nitrogen) under anhydrous conditions at about 0° C. to 50°C. (preferably room temperature), and allowing the reactants to interactfor about 1 to 48 hours.

A solid phase sequential coupling procedure can be performed usingestablished methods such as use of an automated peptide synthesizer. Inthis procedure, an amino protected amino acid is bound to a resinsupport at its carboxyl terminus, the protected amine is deprotectedwhere the peptide linkage is desired, the amino group neutralized with abase and the next amino protected amino acid in the desired sequence iscoupled in a peptide linkage. The deprotection, neutralization andcoupling steps are repeated until the desired peptide is synthesized.The compounds provided herein are thus synthesized from their carboxylterminal end to their amino terminal end. The amino protected amino acidcan be a conventional amino acid, a derivative or isomer thereof, or aspacer group. The resin support employed can be any suitable resinconventionally employed in the art for the solid phase preparation ofpolypeptides. The preferred resin is polystyrene which has beencross-linked with from about 0.5 to about 3% divinyl benzene, which hasbeen either benzhydrylamidated, chloromethylated or hydroxymethylated toprovide sites for amide or ester formation with the initially introducedamino protected amino acid.

An example of a hydroxymethyl resin is described by Bodansky et al.[Chem. Ind. (London) 38, 1597-98 (1966)]. The preparation ofchloromethyl and benzhydrylamine resins are described by Stewart et al.["Solid Phase Peptide Synthesis," 2nd Edition, Pierce Chemical Co.,Rockford, Ill. (1984). Chapter 2, pp. 54-55]. Many of these resins areavailable commercially. In general, the amino protected amino acid whichis desired on the carboxyl-terminal end of the peptide is bound to theresin using standard procedures and practices as are well known andappreciated in the art. For example, the amino protected amino acid canbe bound to the resin by the procedure of Gisin [Helv. Chem. Acta, 56,1476 (1973)]. When it is desired to use a resin containing abenzhydrylamine moiety as the resin binding site an amino protectedamino acid is coupled to the resin through an amide linkage between itsα-carboxylic acid and the amino moiety of the resin. The coupling iseffected using standard coupling procedures as described below. Manyresin-bound amino acids are available commercially.

The α-amino protecting group employed with each amino acid introducedinto the polypeptide sequence may be any such protecting group known inthe art. Among the classes of amino protecting groups contemplated are:(1) acyl type protecting groups such as formyl, trifluoroacetyl,phthalyl, p-toluenesulfonyl (tosyl), benzenesulfonyl,nitrophenylsulfonyl, tritylsulfonyl, o-nitrophenoxyacetyl, andα-chlorobutryl; (2) aromatic urethane type protecting groups such asbenzyloxycarbonyl and substituted benzyloxycarbonyls such asp-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, and benzhydryloxycarbonyl;(3) aliphatic urethane protecting groups such as t-butyloxycarbonyl(BOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,and allyloxycarbonyl; (4) cycloalkyl urethane type protecting groupssuch as cyclopentyloxycarbonyl, adamantyloxycarbonyl, andcyclohexyloxycarbonyl; (5) thiourethane type protecting groups such asphenylthiocarbonyl; (6) alkyl type protecting groups such astriphenylmethyl (trityl) and benzyl (Bn); and (7) trialkylsilaneprotecting groups such as trimethylsilane, 4-[-(4-chlorophenyl)sulfonylaminocarbonyl] phenyl carbonyl, and 4-[-(4-bromophenyl)sulfonylaminocarbonyl] phenyl carbonyl. The preferred α-amino protectinggroup is t-butyloxycarbonyl (BOC); its use as an α-amino protectinggroup for amino acids is well known to those of skill in the art ([see,e.g., by Bodansky et al. in "The Practice of Peptide Synthesis,"Springer-Verlag, Berlin (1984), p.20].

Following the coupling of the amino protected amino acid to the resinsupport, the α-amino protecting group may be removed using any suitableprocedure such as by using trifluoroacetic acid, trifluoroacetic acid inCH₂ Cl₂, or HCl in dioxane. The deprotection is carried out at atemperature of between 0° C. and room temperature. Other standardcleaving reagents may be used for removal of specific amino protectinggroups under conditions well known and appreciated in the art.

After removal and neutralization of the α-amino protecting group, thenext desired amino-protected amino acid is coupled through a peptidelinkage. This deprotection, neutralization and coupling procedure isrepeated until a peptide of the desired sequence is obtained.Alternatively, multiple amino acid groups may be coupled by the solutionmethod prior to coupling with the resin supported amino acid sequence.

The selection and use of an appropriate coupling reagent is within theskill of the skilled artisan. Particularly suitable coupling reagentswhere the amino acid to be added is Gln, Asn, or Arg includeN,N-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole. The use ofthese reagents prevents nitrile and lactam formation. Other couplingagents are (1) other carbodiimides (e.g.,N-ethyl-N'-(y-dimethylaminopropylcarbodiimide); (2) ketenimines; (3)isoxazolium salts (e.g., N-ethyl-5-phenylisoxazolium-3-sulfonate); (4)monocyclic nitrogen-containing heterocyclic amides of aromatic charactercontaining one through four nitrogens in the ring such as imidazolides,pyrazolides, and 1,2,4-triazolides (specific heterocyclic amides thatare useful include N,N-carbonyldiimidazole andN,N-carbonyl-di-1,2,4-triazole); (5) alkoxylated acetylene (e.g.,ethoxyacetylene); (6) reagents which form a mixed anhydride with thecarboxyl moiety of the amino acid (e.g., ethyl chloroformate andiso-butyl chloroformate) or the symmetrical anhydride of the amino acidto be coupled (e.g., BOC-Ala-O-Ala-BOC); and (7) nitrogen-containingheterocyclic compounds having a hydroxyl group on one ring nitrogen(such as, but not limited to, N-hydroxyphthalimide,N-hydroxysuccinimide, and 1-hydroxybenzotriazole). Other activatingreagents and their use in peptide coupling are described by Kapoor [J.Pharm. Sci., 15, 1-27 (1970)]. Use of the symmetrical anhydride as thecoupling agent is the generally preferred amino acid coupling methodherein.

The preferred coupling method for Gin, Asn and Arg is to react theprotected amino acid, or derivatives or isomers thereof, withN,N-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (1:1) in DMF inthe presence of the resin or resin-bound amino acid or peptide. Thepreferred coupling method for other amino acids involves reacting theprotected amino acid, or derivative or isomer thereof, withN,N-dicyclohexylcarbodiimide in CH₂ Cl₂ to form the symmetricalanhydride. The symmetrical anhydride is then introduced into the solidphase reactor containing the resin or resin-bound amino acid or peptide,and the coupling is carried out in a medium of DMF, or CH₂ Cl₂, or DMF:CH₂ Cl₂ (1:1). A medium of DMF is preferred. The success of the couplingreaction at each stage of the synthesis is monitored by a ninhydrin testas described by Kaiser et al. [Analyt. Biochem. 34, 595 (1970)]. Incases where incomplete coupling occurs, the coupling procedure isrepeated. If the coupling is still incomplete, the deprotected amine iscapped with a suitable capping reagent to prevent its continuedsynthesis. Suitable capping reagents and the use thereof are well knownand appreciated in the art. Examples of suitable capping reagents areacetic anhydride and acetylimidazole as described by Stewart et al.["Solid Phase Peptide Synthesis," 2nd Ed., Pierce Chemical Co.,Rockford, Ill. (1984), Chapter 2, p.73].

After the desired amino acid sequence has been obtained, the peptide iscleaved from the resin. This can be effected by procedures which arewell known and appreciated in the art, such as by hydrolysis of theester or amide linkage to the resin. It is preferred to cleave thepeptide from the benzhydrylamine resin with a solution of dimethylsulfide, p-cresol, thiocresol, or anisole in anhydrous hydrogenfluoride. The cleavage reaction is preferably carried out attemperatures between about 0° C. and about room temperature, and isallowed to continue preferably from between about 5 minutes to about 5hours.

As is known in the art of solid phase peptide synthesis, many of theamino acids bear side chain functionalities requiring protection duringthe preparation of the peptide. The selection and use of an appropriateprotecting group for these side chain functionalities is within theability of those skilled in the art and will depend upon the amino acidto be protected and the presence of other protected amino acid residuesin the peptide. The selection of such a side chain protection group iscritical in that it must not be removed during the deprotection andcoupling steps of the synthesis. For example, when BOC is used as theα-amino protecting group, the following side chain protecting groups aresuitable: p-toluenesulfonyl (tosyl) moieties can be used to protect theamino side chains of amino acids such as Lys and Arg; p-methylbenzyl,acetamidomethyl, benzyl (Bn), or t-butylsulfonyl moieties can be used toprotect the sulfide-containing side chains of amino acids such ascysteine, homocysteine, penicillamine and the like or derivativesthereof; benzyl or cyclohexyl ester moieties can be used to protectcarboxylic acid side chains of amino acids such as Asp, Glu; a benzylether can be used to protect the hydroxyl-containing side chains ofamino acids such as Ser and Thr; and a 2-bromocarbobenzoxy (2Br-Cbz)moiety can be used to protect the hydroxyl-containing side chains ofamino acids such as Tyr. These side chain protecting groups are addedand removed according to standard practices and procedures well known inthe art. It is preferred to deprotect these side chain protecting groupswith a solution of anisole in anhydrous hydrogen fluoride (1:10).Typically, deprotection of side chain protecting groups is performedafter the peptide chain synthesis is complete but these groups canalternatively be removed at any other appropriate time. It is preferredto deprotect these side chains at the same time as the peptide iscleaved from the resin.

The compounds are then isolated and purified by standard techniques. Thedesired amino acids, derivatives and isomers thereof can be obtainedcommercially or can be synthesized according to standard practices andprocedures well known in the art.

C. Identification of preferred compounds using assays that identifycompounds that modulate processing of amyloid precursor protein (APP) orthat modulate other selected processing pathways

Compounds provided herein modulate the processing of proteins, such asamyloid precursor protein (APP), involved in diseases. Particularcompounds may be selected for treatment of a particular disorderempirically using in vitro or in vivo animal models, such as those thatwere used to identify the ketones and aldehyde compounds that correspondto the alcohols provided herein.

For example, among the compounds provided herein are those that modulateprocessing of APP. This modulation can be demonstrated in a variety ofways. For example, compounds can be evaluated for the ability tomodulate generation of Aβ or α-sAPP.

1. In vitro assays

The compounds provided herein yield a positive result in one or more invitro assays that assess the effects of test compounds on processing ofAPP. In particular, in vitro assay systems for identifying suchcompounds are provided herein. These assays evaluate the effects of atest compound on processing of APP and use cultured human glioblastomacell lines that have been transfected with DNA encoding either awild-type 695 amino acid isoform of APP or a mutein of APP that containschanges (in this case two or three amino acid changes have been made)that appear to make the molecule more susceptible to proteolyticcleavage that results in increased production of Aβ [see, e.g., Mullanet al. (1992) Nature Genet. 1:345-347].

In performing these assays, a test compound is added to the culturemedium and, after a selected period of time, the culture medium and/orcell lysates are analyzed using immunochemical assays to detect therelative amounts of Aβ, total soluble APP (sAPP), a portion of sAPPdesignated α-sAPP, and C-terminal fragments of APP. In particular, theculture medium and cell lysates are analyzed by immunoblotting coupledwith laser scanning densitometry and ELISAs using several differentantibodies. A positive test, occurs when: (1) there is a decrease in the˜4-kDa amyloid β-protein (Aβ) in the medium relative to control cultures(4-kDa assay); and/or (2) the relative amount of sAPP in the mediumincreases; and/or (3) there is a decrease in the amount of C-terminalamyloidogenic fragments larger than 9 kDa and smaller than 22 kDa in thecell lysate as a result of differential processing; and/or (4) there isan increase in the amount of α-sAPP in the medium relative to controlcultures. Control cultures can be cultures that have not been contactedwith the compound. The Aβ assay is done using cells (e.g., HGB 717/Swed)that have been transfected with DNA encoding the mutein APP; the otherassays are performed using cells, such as HGB695 cells, that have beentransfected with DNA encoding a wild-type APP.

Preferred compounds have activity that is at least 2-fold, preferably5-fold, more preferably 10-fold, most preferably 50-100-fold, greateractivity than N-Acetylleucylleucyinorleucinal [see, e.g., EP 0 504 938A2; and Sherwood et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:3353-3357] in at least one, preferably the Aβ assay, of these assays.

2. The amount of α-sAPP and the ratio of α-sAPP to total sAPP incerebral spinal fluid (CSF) as an indicator of Alzheimer's Disease (AD)and the effectiveness of therapeutic intervention

The relative amount of α-sAPP and the ratio of α-sAPP to total sAPP inCSF are shown herein to be useful markers in the detection ofneurodegenerative disorders characterized by cerebral deposition ofamyloid (e.g., AD) and in monitoring the progression of such disease.Furthermore, assay systems incorporating these markers can be used inmonitoring therapeutic intervention of these diseases.

The amount of α-sAPP and the ratio of α-sAPP to total sAPP in CSFsamples can be used as an indicator of Alzheimer's Disease and otherneurodegenerative disorders. For purposes herein, this amount and/or theratio can also be used to assess the effectiveness of compounds providedherein in treating Alzheimer's Disease and neurodegenerative disorders.

It has been found that patients with suspected Alzheimer's disease (asdiagnosed by other indicia, or confirmed by autopsy) have astatistically significant lower ratio of α-sAPP to total sAPP in CSF andalso have statistically significant lower levels of α-sAPP. Therefore,by comparison with non-Alzheimer's disease controls or by existence of aratio lower than a predetermined standard, based, for example, onaverages in samples from large numbers of unafflicted individuals, or anamount of α-sAPP lower than a predetermined standard, Alzheimer'sdisease or, depending upon other indications, another neurodegenerativedisease is indicated.

Compounds, such as those provided herein, that alter this ratio or thelevel of α-sAPP closer to that of individuals who do not have aneurodegenerative disorder characterized by the cerebral deposition ofamyloid are considered useful for treating these disorders.

3. In vivo assays

The ability of compounds to modulate processing of APP can also beevaulated using in vivo assays [see, e.g., Lamb et al. (1993) NatureGenet. 5:22-29; Pearson et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:10578-10582; Kowall et al. (1991) Proc. Natl. Acad. Sci. U.S.A.88:7247-7251]. Compounds can be administered through a canula implantedin the cranium of a rat or other suitable test animal. After apredetermined period of administration the rats are sacrificed. Thehippocampi are evaluated in immunoblot assays or other suitable assaysto determine the relative level of α-sAPP and C-terminal fragments ofAPP compared to untreated control animals. Compounds that result inrelative increases in the amount of α-sAPP are selected.

D. Formulation of pharmaceutical compositions

Compositions are provided that contain therapeutically effective amountsof the compounds of formulae (I), (II) and (III). The compounds arepreferably formulated into suitable pharmaceutical preparations such astablets, capsules or elixirs, for oral administration or in sterilesolutions or suspensions for parenteral administration, as well astransdermal patch preparation. Typically the compounds described aboveare formulated into pharmaceutical compositions using techniques andprocedures well known in the art.

About 10 to 500 mg of a compound or mixture of compounds for Formulae(I), (II) and (III) or a physiologically acceptable salt is compoundedwith a physiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavor, etc., in a unit dosage form as calledfor by accepted pharmaceutical practice. The amount of active substancein those compositions or preparations is such that a suitable dosage inthe range indicated is obtained.

To prepare compositions, one or more compounds of formulae (I), (II) and(III) are mixed with a suitable pharmaceutically acceptable carrier.Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. Liposomal suspensions mayalso be suitable as pharmaceutically acceptable carriers. These may beprepared according to methods known to those skilled in the art. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. The effective concentrationis sufficient for ameliorating the symptoms of the disease, disorder orcondition treated and may be empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. In addition, the active materials can also be mixed withother active materials that do not impair the desired action, or withmaterials that supplement the desired action or have other action. Thecompounds may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as tween, or dissolution in aqueous sodium bicarbonate. Derivativesof the compounds, such as salts of the compounds or prodrugs of thecompounds may also be used in formulating effective pharmaceuticalcompositions.

The concentrations or the compounds are effective for delivery of anamount, upon administration, that ameliorates the symptoms of thedisorder for which the compounds are administered. Typically, thecompositions are formulated for single dosage administration.

The compounds of formulae (I), (II) and (III) may be prepared withcarriers that protect them against rapid elimination from the body, suchas time release formulations or coatings. Such carriers includecontrolled release formulations, such as, but not limited to,microencapsulated delivery systems.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in known in vitro and in vivo model systems forthe treated disorder.

The compositions can be enclosed in ampules, disposable syringes ormultiple or single dose vials made of glass, plastic or other suitablematerial. Such enclosed compositions can be provided in kits.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the active compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder,such as, but not limited to, gum tragacanth, acacia, corn starch orgelatin; an excipient such as microcrystalline cellulose, starch andlactose, a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a glidant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, and fruitflavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, a naturally occurring vegetable oil like sesame oil, coconut oil,peanut oil, cottonseed oil, etc. or a synthetic fatty vehicle like ethyloleate or the like, polyethylene glycol, glycerine, propylene glycol orother synthetic solvent; antimicrobial agents, such as benzyl alcoholand methyl parabens; antioxidants, such as ascorbic acid and sodiumbisulfite; chelating agents, such as ethylenediaminetetraacetic acid(EDTA); buffers, such as acetates, citrates and phosphates; and agentsfor the adjustment of tonicity such as sodium chloride or dextrose.Parenteral preparations can be enclosed in ampules, disposable syringesor multiple dose vials made of glass, plastic or other suitablematerial. Buffers, preservatives, antioxidants and the like can beincorporated as required.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof. Liposomalsuspensions, including tissue-targeted liposomes, may also be suitableas pharmaceutically acceptable carriers. These may be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.

The active compounds may be prepared with carriers that protect thecompound against rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others. Methodsfor preparation of such formulations are known to those skilled in theart.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Suchsolutions, may be formulated as 0.01%-100% (weight to volume) isotonicsolutions, pH about 5-7, with appropriate salts. The compounds may beformulated as aeorsols for topical application, such as by inhalation[see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923].

Finally, the compounds may be packaged as articles of manufacturecontaining packaging material, an acceptable composition containing acompound of formulae (I), (II) and (III) provided herein, which iseffective for treating the particular disorder, and a label thatindicates that the compound or salt thereof is used for treating thedisorder.

E. Methods of use

The compounds for use in the methods herein have the formulae (I), (II)and (III): ##STR7## or the hydrates and isosteres, diastereomericisomers and mixtures thereof, or pharmaceutically acceptable saltsthereof in which X is selected as described above, and R₁, R₂, R₃, R₄,R₅, R₆, R₇, R₈, R_(A), R_(B), Q and n are selected from among (i), (ii),(iii), (iv), (v), (vi), (vii) or (viii), as described above.

These compounds have pharmacological utility and also utility asreagents. It is recognized in this art that compounds that exhibitactivity as protease inhibitors have use for the treatments ofparticular disorders in which particular proteases are implicated. Forexample compounds that exhibit activities in assays that assess theability of the compounds to alter or modulate the activity of proteinsassociated with the deposition of cerebral amyloid, arepharmacologically useful and potentially therapeutically useful in thetreatment of disorders that involve such deposition.

The dose ranges, which can be established empirically, for use in thetreatment of disease states will depend upon the etiology, nature, andseverity of the disease state as well as such other factors asdetermined by the attending physician. The broad range for effectivetreatment is about 0.01 to 10 mg per kilogram of body weight per day.The preferred range is about 0.1 to 10 mg/Kg of body weight per day.

Included among the compounds for use in the methods here in are thosethat are alcohols, preferably secondary alcohols, that correspond topeptidyl or petidyl analog ketone protease inhibitors [see, e.g., Skileset al (1992) J. Med. Chem. 35:641-662; Mjali et al. (1994) Bioorg. Med.CHem. Lttrs. 4:1965-1968; Imperiali et al. (1986) Biochem. 25:3760-3767;Angelastro et al. (1994) J. Med. Chem. 37:4538-4554; published Europeanpatent application EP 0 410 411 A2]. Other compounds are those offormulae (I), (II) and (III).

The active compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, semi-liquid or solid form andare formulated in a manner suitable for each route of administration.Preferred modes of administration include oral and parenteral modes ofadministration.

Since, it is feasible to indirectly measure the presence of, and overthe course of time, to determine the rate of increase of those proteinsegments believed to be critical to the formation of amyloid plaqueslocated in the brain (see, e.g., U.S. Pat. No. 5,270,165 and the CSFassay provided herein and described above and in the EXAMPLES), dosagescan be empirically determined by the physician. As these techniquesinvolve the use of cerebral spinal fluids, such techniques and otherequivalently functioning procedures, will be useful to the attendingphysician in determining the need to modify the dosage for individualpatients.

In treating these disease states, it is sufficient to start treating thepatient as soon as the attending physician makes a diagnosis that thepatient is suffering from one of these diseases. Thus, although theprogress of treatment of the patient may be monitored by themeasurements of those biological factors which characterize thediseases, it is not necessary to so-evaluate such characteristics beforetreatment. Rather it is within the provence of the attending physicianto determine when it is in the best interest of the patient to starttreatment. Therefore, patients showing increased probabilities of thedisease state, (e.g. by carrying known familial genetic markers thatincrease the probability of the incidence of neurodegenerative diseasesas well as the patient's general behavioral characteristics and otherindicia of these diseases) can be treated by the methods and with thecompositions provided herein.

1. Treatment of neurodegenerative diseases

Amyloid plaques are believed to accompany and/or to be involved in theprocess-responsible for the development and progression of certainneurodegenerative disease states. Without being bound by any theory ofaction, it is believed that the compounds provided herein modulate thegeneration of amyloidogenic peptides to effectuate a beneficial result.Without any intent to limit--or restrict--the compounds and methodsprovided herein to any specific mechanism of action for the end-useapplications, it is believed that the compounds effectuate a modulationof the processing of the amyloid precursor protein (APP), the progenitorof the deposited amyloidogenic Aβ peptides (39 to 43 amino acidresidues) found in senile plaques in the brains of patients diagnosedwith, for example, Alzheimer's disease. Thus, the compounds providedherein are useful in the treatment of such neurodegenerative diseasestates in which such amyloid plaques accumulate or are implicated in theetiology thereof, including, but not limited to: Alzheimer's disease,cognition deficits, Down's Syndrome, Parkinson's disease, cerebralhemorrhage with amyloidosis, dementia pugilistica, head trauma and inthe treatment of conditions characterized by a degradation of theneuronal cytoskeleton resulting from a thrombolytic or hemorrhagicstroke.

For example, it is believed that the compounds can be used in thetreatment of Alzheimer's patients through the modulation of APPprocessing to effectuate a beneficial result by: (a) decreasing theformation of Aβ; (b) modulating the generation of a mutually exclusive,alternative-processed form of APP that precludes Aβ formation (α-sAPP);and/or, (c) modulating the generation of partially processed C-terminalAβ-containing amyloidogenic peptides.

In addition, these compounds may also beneficially modulateneurodegenerative abnormalities not thought to be associated withamyloid plaques, such as stroke, by beneficially affecting the rate ofdegeneration of the neuronal cytoskeleton that occurs as a result ofthrombolytic or hemorrhagic stroke.

It is believed that the treatment of patients with such disorders withthese compounds will result in a beneficial modulation of the symptomsof or causative factors involved in neurodegenerative disease states andwill result in an enhanced lifestyle as well as to delay or obviate theneed to institutionalize these patients.

The compounds can be administered to patients in need of such treatmentin a dosage range of 0.01-10 mg per kg of body weight per day. and canbe administered by any appropriate route, for example, orally,parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid, semi-liquid or solid form and are formulated in amanner suitable for each route of administration. As stated above, thedose will vary depending on severity of disease, weight of patient andother factors which a person skilled in the art will recognize.

Patients include those with a neurodegenerative disease, including butnot limited to Alzheimer's disease, cognition deficits, Down's Syndrome,Parkinson's disease, cerebral hemorrhage with amyloidosis, dementiapugilistica, and head trauma. Treatment is effected by administering tosuch patient a therapeutically effective amount of a compound of theformulae (I), (II) and (III) defined as above. Particularly preferredfor use in these methods are the compounds particularly provided herein,including the compounds of formulae (I), (II) and (III) as definedabove, but with the proviso that: (1) at least one of the amino acidresidues in the resulting di or tri-peptide is a non-naturally-occurringα-amino acid or at least one of R₁, R₃ and R₅ is not a side chain of anaturally-occurring amino acid; and (2) when R₁ is the side chain from anon-naturally occurring amino acid and X is a tertiary or secondaryhaloalkyl alcohol, R₁ is not the side chain of cyclohexylalanine orcyclohexylglycine.

In other embodiments, the methods use compounds that have formula (III),as defined above, then, when X is a tertiary or secondary haloalkylalcohol, R₁ is the side chain of a non-naturally-occurring α-amino acidand it is not the side chain of cyclohexylalanine or cyclohexylglycine.

In certain other embodiments, the methods use compounds that haveformulae (I) or (II), as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-occurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occurringamino acid, R₁ is not the side chain of cyclohexylalanine orcyclohexylglycine.

In certain other embodiments, the methods use compounds that haveformulae (I), (II) or (III) as defined above, but with the proviso that,when the compounds have formula (I) or (II): R₁ is a subunit of anon-naturally-occurring amino acid, the side chain of R₁ is of anon-naturally-occurring amino acid other than cyclohexylalanine orcyclohexylglycine, unless the compounds are primary alcohols, then thenon-naturally-occurring amino acid is other than norleucine ornorvaline.

Thus, in certain other embodiments the methods use compounds that areprimary alcohols, when the compounds are primary alcohols, they haveformulae (I) or (II), particularly formula I, as defined above, but withthe proviso that: (1) at least one of the amino acid residues in theresulting di or tri-peptide is a non-naturally-occurring α-amino acid orat least one of the R₁, R₃ and R₅ is not a side chain of anaturally-occurring amino acid; and (2) when R₁ is the side chain from anon-naturally occurring amino acid, it is not the side chain ofnorleucine or norvaline.

Thus, in certain other embodiments in which the compounds used in themethods are primary alcohols, the compounds have formulae (I) or (II),particularly formula I, as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-occurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occurringamino acid, it is not the side chain of cyclohexylalanine,cyclohexylglycine, norleucine or norvaline.

In other embodiments, the methods use compouds that have formulae (I),(II) or (III) as defined above, but with the proviso that, when thecompounds have formula (I) or (II): R₁ is a side chain of anon-naturally-occurring amino acid other than cyclohexylalanine orcyclohexylglycine, norleucine or norvaline.

In other embodiments, the methods use compounds that have formulae (I),(II) or (III) as defined above, but with the proviso that, when thecompounds have formula (I) or (II): R₁ is a side chain of anon-naturally-occurring amino acid other than cyclohexylalanine orcyclohexylglycine, norleucine, norvaline, citrulline, ornithine,4-phenyl-2-aminobutyric acid, 1-naphthylalanine, 2-naphthylalanine,sarcosine, 2-indolinecarboxylic acid, β-alanine, β-valine,N-6-acetyllysine, O-4'-methyltyrosine, a substituted alanine andguanidinophenylalanine.

In certain other embodiments, the methods use compounds that haveformulae (I), (II) or (III) as defined above, but with the proviso that,when the compounds have formula (I) or (II): at least one of the aminoacid residues in the resulting di-peptide or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₁, preferably R₁, is a side chain of a non-naturally-occurring aminoacid, R₁ is not cyclohexylalanine, and the at least one non-naturallyoccurring amino acid (or side chain thereof) is other than norleucine ornorvaline, unless the resulting residue is a halo-substituted alcohol,particularly fluoro-substituted alcohols. Such compounds include, butare not limited to: (2SR)-(3SR)-N-Ac-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide and (2SR)-(3SR)-N-valeroyl-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide.

In other embodiments, the methods use compounds that have formulae (I)or (II) and at least one of R₁, R₃ and R₅, preferably R₁ or R₅, includesat least one unsaturated bond so that at least one of R₁, R₃ and R₅,preferably R₁ and R₅, is a straight or branched carbon chain containingat least one unsaturated bond, preferably a double bond, and 2 to 10,preferably 3 to 7, more preferably 4 to 6, carbon atoms in the chain.Such side chains include, but are not limited to substituted andunsubstituted propenes, butenes, pentenes, such as, 2-methyl-propenyland 2-butenyl, which are among the preferred residues.

2. Treatment of diseases characterized by degeneration of thecytoskeleton

Also provided are methods of treating a patient suffering from a diseasestate characterized by the degeneration of the cytoskeleton arising froma thrombolytic or hemorrhagic stroke by administering a therapeuticallyeffective amount of a compound of the formulae (I), (II) or (III)defined as above.

Particularly preferred for use in these methods are the compoundsparticularly provided herein, including the compounds of formulae (I),(II) and (III) as defined above, but with the proviso that: (1) at leastone of the amino acid residues in the resulting di or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-occurring amino acid; and (2) whenR₁ is the side chain from a non-naturally occurring amino acid and X isa tertiary or secondary haloalkyl alcohol, R₁ is not the side chain ofcyclohexylalanine or cyclohexylglycine.

In other embodiments, the methods use compounds that have formula (III),as defined above, then, when X is a tertiary or secondary halaloalkylalcohol, R₁ is the side chain of a non-naturally-occurring α-amino acidand it is not the side chain of cyclohexylalanine or cyclohexylglycine.

In certain other embodiments, the methods used compounds that haveformulae (I) or (II), as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-occurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occurringamino acid, R₁ is not the side chain of cyclohexylalanine orcyclohexylglycine.

In certain other embodiments, the methods use compounds that haveformulae (I), (II) or (III) as defined above, but with the proviso that,when the compounds have formula (I) or (II): R₁ is a subunit of anon-naturally-occurring amino acid, the side chain of R₁ is of anon-naturally-occurring amino acid other than cyclohexylalanine orcyclohexylglycine, unless the compounds are primary alcohols, then thenon-naturally-occurring amino acid is other than norleucine ornorvaline.

Thus, in certain other embodiments the methods use compounds that areprimary alcohols, when the compounds are primary alcohols, they haveformulae (I) or (II), particularly formula I, as defined above, but withthe proviso that: (1) at least one of the amino acid residues in theresulting di or tri-peptide is a non-naturally-occurring α-amino acid orat least one of the R₁, R₃ and R₅ is not a side chain of anaturally-occurring amino acid; and (2) when R₁ is the side chain from anon-naturally occurring amino acid, it is not the side chain ofnorleucine or norvaline.

Thus, in certain other embodiments in which the compounds used in themethods are primary alcohols, the compounds have formulae (I) or (II),particularly formula I, as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-occurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occurringamino acid, it is not the side chain of cyclohexylalanine,cyclohexylglycine, norleucine or norvaline.

In other embodiments, the methods use compouds that have formulae (I),(II) or (III) as defined above, but with the proviso that, when thecompounds have formula (I) or (II): R₁ is a side chain of anon-naturally-occurring amino acid other than cyclohexylalanine orcyclohexylglycine, norleucine or norvaline.

In other embodiments, the methods use compounds that have formulae (I),(II) or (III) as defined above, but with the proviso that, when thecompounds have formula (I) or (II): R₁ is a side chain of anon-naturally-occurring amino acid other than cyclohexylalanine orcyclohexylglycine, norleucine, norvaline, citrulline, ornithine,4-phenyl-2-aminobutyric acid, 1-naphthylalanine, 2-naphthylalanine,sarcosine, 2-indolinecarboxylic acid, β-alanine, β-valine,N-6-acetyllysine, O-4'-methyltyrosine, a substituted alanine andguanidinophenylalanine.

In certain other embodiments, the methods use compounds that haveformulae (I), (II) or (III) as defined above, but with the proviso that,when the compounds have formula (I) or (II): at least one of the aminoacid residues in the resulting di-peptide or tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅, preferably R₁, is a side chain of a non-naturally-occurring aminoacid, R₁ is not cyclohexylalanine, and the at least one non-naturallyoccurring amino acid (or side chain thereof) is other than norleucine ornorvaline, unless the resulting residue is a halo-substituted alcohol,particularly fluoro-substituted alcohols. Such compounds include, butare not limited to: (2SR)-(3SR)-N-Ac-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide and (2SR)-(3SR)-N-valeroyl-L-LeuN-(3-(1,1,1-trifluoro-2-heptanol)]amide.

In other embodiments, the methods use compounds that have formulae (I)or (II) and at least one of R₁, R₃ and R₅, preferably R₁ or R₅, includesat least one unsaturated bond so that at least one of R₁, R₃ and R₅,preferably R₁ and R₅, is a straight or branched carbon chain containingat least one unsaturated bond, preferably a double bond, and 2 to 10,preferably 3 to 7, more preferably 4 to 6, carbon atoms in the chain.Such side chains include, but are not limited to substituted andunsubstituted propenes, butenes, pentenes, such as, 2-methyl-propenyland 2-butenyl, which among the preferred residues.

3. Protease inhibition in cells

The compounds provided herein have activity as inhibitors of cellularproteases, such as cysteine proteases, including calpain. It is believedby those of skill in this art that excessive activation of the Ca²⁺-dependent protease calpain plays a role in the pathology of a varietyof disorders, including cerebral ischaemia, cataract, myocardialischaemia, muscular dystrophy and platelet aggregation. Thus, compoundsthat have activity as calpain inhibitors are considered by those ofskill in this art to be useful [see, e.g., U.S. Pat. No. 5,081,284,Sherwood et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3353-3357].Assays that measure the anti-calpain activity of selected compounds areknown to those of skill in the art (see, e.g., U.S. Pat. No. 5,081,284).Activities of inhibitors in such in vitro assays at concentrations(IC₅₀) in the nanomolar range or lower are indicative of therapeuticactivity. Such compounds also have utility in the purification ofproteinases, such as cysteine proteases, on affinity columns of thesecompounds (see, U.S. Pat. No. 5,081,284). Also, calpain inhibitors, suchas N-Acetylleucylleucyinorleucinal [see, e.g., EP 0 504 938 A2; andSherwood et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3353-3357],which is commercially available, are used as reagents in the study ofprotein trafficking and other cellular processes [see, e.g., Sharma etal. (1992) J. Biol. Chem. 2671:5731-5734]. Finally, inhibitors ofcysteine proteases strongly inhibit the growth of Plasmodium falciparumand Schistosoma mansoni [see, e.g., Scheibel et al. (1984) Proteaseinhibitors and antimalarial effects. In: Malaria and the Red Cell,Progress in Clinical and Biological Research, Alan R. Liss, Inc., NY,pp. 131-142]. Thus, the compounds herein may be used as such reagents orto inhibit the growth of certain parasites.

The methods of protease inhibition in cells may employ any of thecompounds of formulae (I), (II) and (III) or peptidyl, peptidyl analogand amino acid analog alcohols, particularly haloalkyl secondaryalcohols that are the corresponding alcohols of any peptidyl or peptidylanalog ketones or aldehydes that inhibit proteases in cell-free assays[see, EP 0 410 411 A2, which is based on U.S. application Ser. No.07/385,624, WO 92/20357, which is based on U.S. application Ser. No.08/704,449, EP 0 364 344 A2, which is based on U.S. application Ser. No.07/254,762]. In practicing these methods, cells are contacted with thecompound. Compounds that have specificity for a particular protease maybe selected by contacting the compounds with cells that producecompounds processed by such protease or that express such protease.Compounds that result in a decrease in such processing or proteaseactivity are selected. The APP processing assay set forth herein isexemplary of such cell-based assays.

The following specific examples further illustrate the methods by whichcompounds of formulae (I), (II) and (III) may be prepared, but are notmeant to limit the scope of this invention to the specific compounds.Thus, the following examples are included for illustrative purposes onlyand are not intended to limit the scope of the invention.

EXAMPLE 1 Preparation of L-(methyl)Nle Methyl Ester Hydrochloride

To a stirred solution of the N-BOC-L-Nle methyl ester (1.0 eq) in 20:1anhydrous tetrahydrofuran:dimethylformamide (THF:DMF) at 0° C. underArgon (Ar) is added methyl iodide (2.0 eq) and 60% sodium hydride (NaH)in oil dispersion (1.1 eq). The reaction mixture is refluxed for 16 h.The mixture is poured onto 10% aqueous hydrogen chloride (10% HCl) andis extracted with ethyl acetate (EA). The combined organic extracts arewashed with saturated aqueous sodium chloride (sat. NaCl), dried overmagnesium sulfate (MgSO₄), filtered and concentrated in vacuo to affordN-BOC-L-(methyl)Nle methyl ester.

N-BOC-L-(methyl)Nle methyl ester (1.0 eq) is treated with 4 N hydrogenchloride (4 N HCl) in dioxane at room temperature (R.T.) The reactionmixture is stirred for 1.5 h then concentrated in vacuo. The solid istreated with anhydrous ether and concentrated in vacuo. The resultingL-(methyl)Nle methyl ester hydrochloride is isolated.

EXAMPLE 2 Preparation of the Precusor Ethyl2-amino-4-methyl-4-pentenoate hydrochloride

To a solution of N-(diphenylmethylene)glycine ethyl ester (6.6 g, 24.7mmol) in anhydrous THF at -78° C. under Ar was slowly added 1.0 Mlithium bis(trimethylsilyl)amide (LiHMDSi) in THF (24.7 mL, 24.7 mmol)over 15 min. Stirring was continued for 30 min at -78° C., then3-bromo-2-methylpropene (2.5 mL, 25.0 mmol) was added. The mixture wasgradually warmed to R.T. then stirred for 1 h at R.T. The reactionmixture was treated with water (H₂ O). and then concentrated. Theresidue was taken up in EA (50 mL). The organic layer was washed withsaturated aqueous sodium chloride (sat. NaCl) (2×10 mL), dried (MgSO₄),filtered and concentrated. The crude was purified by flashchromatography on silica gel ((ethyl acetate:hexane) EA:H; 1:4) to giveethyl 4methyl-2-[(diphenylmethylene)amine]-4-pentenoate as a colorlessoil (6.4 g, 89.3%): ¹ H NMR (CDCl₃, 300 MHz) δ1.23-1.29 (t, 3H, J=6.0Hz), 1.48-1.49 (m, 3H), 2.55-2.69 (m, 2H). 4.11-4.24 (m, 3H), 4.71-4.75(m, 2H), 7.16-7.83 (m, 10 H) ppm.

To a stirred solution of the above ethyl ester (6.4 g, 20.0 mmol) inanhydrous ether (15 mL) at R.T. was added 1 N aqueous hydrogen chloride(1 N HCl) (70 mL). After 40 min, the two phases were separated, and theaqueous layer was washed with ether (3×10 mL). The aqueous layer wasadjusted with 1 N aqueous sodium hydroxide (1 N NaOH) (pH=10), thenextracted with ether (3×20 mL). The combined organic extracts were dried(MgSO₄), filtered and then adjusted with 4 N HCl/dioxane (pH =3) andconcentrated in vacuo to afford ethyl 2-amino-4-methyl-4-pentenoatehydrochloride as an oil (3.11 g, 79.4%): ¹ H NMR (CDCl₃, 300 MHz)δ1.25-1.32 (t, 3H, J=6.0 Hz), 1.81 (s, 3 H), 2.74-2.81 (m, 2H),4.22-4.29 (m, 2H), 4.98 (d, 2H, J=12.0 Hz) ppm.

EXAMPLE 3 Preparation of (2SR)-N-Cbz-L-Leu-L-LeuN-[2-(4-methyl-4-pentenol)]amide

To a stirred solution of N-Cbz-L-Leu-OH (5.6 g, 20.7 mmol) in anhydrousmethylene chloride (CH₂ Cl₂) (50 mL) at R.T. under Ar were addedsuccessively hydrobenzotriazole hydrate (HOBT) (5.6 g, 41.5 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (4.0g, 20.7 mmol), H-L-Leu-OCH₃ HCl (3.4 g, 18.8 mmol) and triethylamine(Et₃ N) (2.9 mL, 20.7 mmol). The reaction mixture was stirred for 16 h.The mixture was taken up in additional CH₂ Cl₂ (30 mL), washed with sat.NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated. The resultingresidue was purified by flash chromatography on silica gel (EA:H; 1:2)to afford the dipeptide N-Cbz-L-Leu-L-Leu-OCH₃ as a white solid (6.0 g,64.1%): ¹ H NMR (CDCl₃, 300 MHz) δ0.86-0.95 (ol-t, 12H, J=6.0 Hz),1.48-1.74 (m, 6H), 3.73 (s, 3H), 4.20-4.25 (m, 1H), 4.56-4.63 (m, 1H),5.11 (s, 2H), 5.20 (d, 1H, J=6.0 Hz), 6.36 (d, 1H, J=6.0 Hz), 7.31-7.39(m, 5H) ppm.

To a stirred solution of N-Cbz-L-Leu-L-Leu-OCH₃ (6.0 g, 14.6 mmol) inMeOH/H₂ O (3:1) (60 mL) at R.T. was added lithium hydroxide monohydrate(LiOH.H₂ O) (1.0 g, 43.9 mmol) and hydrogen peroxide (H₂ O₂) (30% weightin H₂ O) (4.5 mL, 43.9 mmol). The reaction mixture was stirred for 3.5 hand then quenched with 10% HCl. The resulting mixture was extracted withEA (3×20 mL). The combined organic extracts were washed with sat. NaCl(2×10 mL), dried (MgSO₄) and concentrated in vacuo to affordN-Cbz-L-Leu-L-Leu-OH as a white solid (5.0 g, 90.5%): ¹ H NMR (CDCl₃,300 MHz) δ0.89-0.92 (t, 12H, J=3.0 Hz), 1.48-1.71 (m, 6H), 4.11 -4.13(m, 1H), 4.55 -4.62 (m, 1H), 5.09 (s, 2H), 5.64 (d, 1H, J=9.0 Hz), 6.83(d, 1H, J=9.0 Hz), 7.33-7.40 (m, 5H) ppm.

To a stirred solution of N-Cbz-L-Leu-L-Leu-OH (3.2 g, 8.5 mmol) inanhydrous CH₂ Cl₂ (35 mL) at R.T. under Ar were added HOBT (2.3 g, 17.1mmol), EDC (1.6 g, 8.5 mmol), ethyl 2-amino-4-methyl-4-pentenoatehydrochloride, as prepared in example 2, (1.5 g, 7.8 mmol) and Et₃ N(1.2 mL, 8.5 mmol). The reaction mixture was stirred for 16 h at R.T.The mixture was taken up in CH₂ Cl₂ (30 mL). The organic layer waswashed with saturated aqueous sodium hydrogen carbonate (sat. NaHCO₃)(2×10 mL), 10% HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated. The residue was purified by flashchromatography on silica gel (EA:H; 1:4) to afford(2SR)-N-Cbz-L-Leu-L-Leu N-[2-[ethyl(4-methyl-4-pentenoate)]amide as awhite solid (1.9 g, 47.3%): Reporting a mixture of diastereomers ¹ H NMR(CDCl₃, 300 MHz) δ0.88-0.92 (ol-m, 12H), 1.21-1.29 (ol-m, 3H), 1.51-1.71(ol-m, 9H), 2.39-2.52 (ol-m, 2H), 4.13-4.21 (ol-m, 3H), 4.47-4.82 (ol-m,4H), 5.10 (ol-m, 2H), 5.32 (d, 1H, J=9.0 Hz), 6,44-6.77 (m, 2H),7.29-7.36 (ol-m, 5H) ppm.

To a stirred solution of the above ethyl ester (1.9 g, 3.6 mmol) inanhydrous THF (10 mL) at 0° C. under Ar was added lithium borohydride(LiBH₄) (0.16 g, 7.1 mmol). Stirring was continued for 30 min at 0° C.then the mixture was warmed to R.T. After 1 h, 1 N HCl (1 mL) was addedto the reaction mixture, and then extracted with EA (3×20 mL). Thecombined organic extracts were washed with sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated in vacuo to afford the crude residue.The residue was purified by flash chromatography on silica gel (EA:H;1:3) to yield the title compound as a white solid (1.4 g, 85.6%):Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.88-0.95(ol-m, 12H), 1.47-1.73 (ol-m, 9H), 2.16-2.27 (ol-m, 2H), 3.54-3.67(ol-m, 2H), 4.12 (ol-m, 2H), 4.33-4.40 (ol-m, 1H), 4.74-4.80 (ol-m, 2H),5.03-5.37 (ol-m, 3H), 6.34 -6.37 (ol-m, 2H), 7.30-7.40 (ol-m, 5H) ppm.

EXAMPLE 4 Preparation of (1SR)-N-Cbz-L-Leu-L-LeuN-[2-(thiazole-hexanol)]amide

To a stirred solution of thiazole (0.11 mL, 1.55 mmol) in anhydrousether (8 mL) at -78° C. under Ar was slowly added 1.8 M N-butyl lithiumin hexanes (nBuLi) (1.0 mL, 1.7 mmol). After an additional 20 min ofstirring at -78° C. N-BOC-L-Nle N-methoxy-N-methylamide (0.17 g, 0.62mmol) in anhydrous ether (5 mL) was added. Stirring was continued for 1h at -78° C. then gradually warmed to R.T. The resulting mixture wastreated with 1 N HCl (1 mL), 1 N NaOH (pH =9), and extracted with ether(3×10 mL). The combined organic layers were washed with sat. NaHCO₃(2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:5) afforded the (2S)-N-BOC-2-amino-thiazole-oxo-hexyl derivative as awhite solid (0.14 g, 76%): ¹ H NMR (CDCl₃, 300 MHz) δ0.86 (t, 3H, J=6.0Hz), 1.23-1.62 (m, 13H), 1.66-1.75 (m, 2H), 5.32-5.46 (m, 2H), 7.70 (d;1H, J=3.0 Hz), 8.03 (d, 1H, J=6.0 Hz) ppm.

The above derivative (0.13 g, 0.44 mmol) was treated with 4 NHCl/dioxane (5 mL) at R.T. After 30 min the reaction mixture wasconcentrated in vacuo. The resulting solid was recrystallized (methanol(MeOH)/Ether) to give the hydrochloride as a white solid (0.1 gr, 75%):¹ H NMR (CD₃ OD, 300 MHz) δ0.64-0.73 (t, 3H, J=6.0 Hz), 1.17-1.21 (m,4H), 1.76-1.99 (m, 2H), 4.86-4.91 (m, 1H), 7.72-7.98 (m, 2H) ppm.

To the resulting hydrochloride (0.09 g, 0.33 mmol) in anhydrous CH₂ Cl₂(10 mL) at R.T. under Ar was added HOBT (0.1 g, 0.73 mmol), EDC (0.07 g,0.36 mmol), N-Cbz-L-Leu-L-Leu-OH (0.09 g, 0.33 mmol) and Et₃ N (0.09 mL,0.66 mmol). After 16 h CH₂ Cl₂ (20 mL) was added, and the organic layerwas washed with sat. NaHCO₃ (2×10 mL), 1 N HCl (2×10 mL), sat. NaCl(2×10 mL), dried (MgSO₄), filtered and concentrated. Purification byflash chromatography on silica gel (EA:H; 1:1) afforded(2S)-N-Cbz-L-Leu-L-Leu N-[2-(thiazole-oxo-hexyl]amide as a white solid(0.16 gr, 88%): ¹ H NMR (CDCl₃, 300 MHz) δ0.83-1.06 (m. 15H), 1.20-2.15(m, 12H), 4.10-4.30 (m, 1H), 4.48-4.61 (m, 1H), 5.11 (s, 2H), 5.15-5.25(m, 1H), 5.60-5.70 (m, 1H), 6.32-6.50 (m, 1H), 7.30-7.40 (m, 5H), 7.70(dd, 1H, J=6.0, 3.0 Hz), 8.05 (dd, 1H, J=6.0, 3.0 Hz) ppm.

To a stirred solution of the above ketone derivative (1.0 eq) in MeOH(50 mL) under Ar at 0° C. is added sodium borohydride (NaBH₄) (1.0mmol). Stirring is continued for 30 min at 0° C., then the mixture iswarmed to R.T. After 1 h, 1 N HCl is added to the reaction mixture, andthen extracted with EA. The combined organic extracts are washed with 1N HCl, sat. NaHCO₃, sat. NaCl, dried (MgSO₄), filtered and concentratedin vacuo to afford the crude residue. The residue is purified by flashchromatography on silica gel to yield the title compound.

EXAMPLE 5 Preparation of (2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol

To a stirred solution of 1-nitropentane (1.0 g, 8.5 mmol) andtrifluoroacetaldehyde ethyl hemiacetal (1.2 mL, 8.5 mL) was addedpotassium carbonate (K₂ CO₃ ) (0.06 g, 0.43 mmol). The reaction mixturewas heated at 60° C. under Ar for 3 h. The mixture was cooled to R.T.,then taken up in EA (50 mL). The organic layer was washed with 1 N HCl(2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentratedto give (2SR)-(3SR)-3-nitro-1,1,1-trifluoro-2-heptanol as a crude oil(1.7 g, 93%): ¹ H NMR (CDCl₃, 300 MHz) δ0.91 (t, 3H, J=7.2 Hz),1.24-1.43 (m, 4H), 2.04-2.11 (m, 2H), 4.08-4.77 (m, 3H) ppm.

To a stirred solution of the nitro-alcohol derivative (1.6 g, 7.4 mmol)in MeOH (10 mL) was added Raney Nickel (0.16 g, 10% by weight). Themixture was placed under 35 psi of hydrogen gas (H₂) for 16 h, and thenwas filtered through celite. The celite was washed with MeOH (3×10 mL).The combined organics were concentrated to give(2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol as an oil (0.8 g, 64.9%):¹ H NMR (CD₃ OD, 300 MHz) δ0.89-0.95 (m, 3H), 1.20-2.10 (m, 6H),3.30-4.40 (m, 4H) ppm.

EXAMPLE 6 Preparation of (2SR)-(3SR)-N-Ac-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide

To a stirred solution of (2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol(0.36 g, 2.14 mmol) (as prepared in example 5) in anhydrous CH₂ Cl₂ (20mL) were added N-Ac-Leu-Leu-OH (0.67 g, 2.4 mmol), HOBT (0.33 g, 2.4mmol), EDC (0.46 g, 2.4 mmol) and Et₃ N (0.33 mL, 2.4 mmol). Thereaction mixture was stirred for 18 h then washed with sat. NaHCO₃ (2×10mL), 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:2) afforded the trifluoromethyl alcohol peptide derivative, the titilecompound, as a white solid (0.85 g, 88.8%): Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.89-0.95 (ol-m, 15H), 1.05-1.90(ol-m, 12H), 1.95 (s, 3H), 3.91-4.50 (ol-m, 4H) ppm.

EXAMPLE 7 Preparation of (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide

To a stirred solution of (2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol(0.9 g, 3.4 mmol) (prepared in example 5) in anhydrous CH₂ Cl₂ (15 mL)at R.T. were added N-Cbz-L-Leu-OH (0.58 g, 3.1 mmol), HOBT (0.92 g, 6.8mmol), EDC (0.65 g, 3.4 mmol) and Et₃ N (0.47 mL, 3.4 mmol). Thereaction mixture was stirred for 18 h then washed with sat. NaHCO₃ (2×10mL), 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:4) afforded the trifluoromethyl alcohol peptide derivative, the titlecompound, as a white solid (1.1 g, 82.6%): Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.88-0.95 (ol-m, 9H), 1.29-1.65(ol-m, 9H), 4.00-4.17 (ol-m, 3H), 4.59-5.46 (m, 4H), 6.42-6.81 (m, 1H),7.29-7.38 (m, 5H) ppm.

EXAMPLE 8 Preparation of N-valeroyl-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide

To a stirred solution of valeric acid (2 mL, 15.7 mmol) in anhydrous CH₂Cl₂ (40 mL) at R.T. were added HOBT (4.3 g, 31.5 mmol), EDC (3.0 g, 15.7mmol), H-L-Leu-OCH₃.HCl (2.6 g, 14.3 mmol) and Et₃ N (2.2 mL, 15.7mmol). The reaction mixture was stirred for 18 h then taken up inadditional CH₂ Cl₂ (20 mL), washed with sat. NaHCO₃ (2×10 mL), 1 N HCl(2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:3) afforded N-valeroyl-L-Leu-OCH₃ as a colorless oil (2.7 g, 77.5%): ¹H NMR (CDCl₃, 300 MHz) 16 0.88-1.03 (m, 12H), 1.50-1.69 (m, 6H),2.20-2.25 (m, 2H), 3.74 (s, 3H), 4.62-4.70 (m, 1H), 5.86-5.89 (d=1H, J=9Hz) ppm.

To a stirred solution of the above methyl ester in MeOH/H₂ O (3:1) (30mL) at R.T. was added LiOH.H₂ O (0.80 g, 33.3 mmol). Reaction mixturewas stirred for 1.5 h, quenched with 1 N HCl (15 mL) then extracted withEA (2×40 mL). The combined organic layers were washed with sat. NaCl(3×15 mL), dried (MgSO₄) and concentrated in vacuo to afford the acid asa white solid (2.4 g, 94.3%): ¹ H NMR (CDCl₃, 300 MHz) δ0.88-0.97 (m,12H), 1.49-1.75 (m, 6H), 2.23-2.25 (m, 2H), 4.58-4.65 (m, 1H), 5.98 (d,1H, J=9 Hz) ppm.

To a stirred solution of the above acid (0.9 g, 3.9 mmol) in anhydrousCH₂ Cl₂ (15 mL) were added successively HOBT (1.1 g, 7.8 mmol), EDC(0.76 g, 3.98 mmol) and (2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol(0.66 g, 3.6 mmol) (prepared as outlined in example 5). The reactionmixture was stirred for 18 h then washed with sat. NaHCO₃ (2×10 mL), 1 NHCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:3) afforded the title compound as a white solid (0.95 g, 67.2%):Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.88-0.95(ol-m, 15H), 1.31-1.83 (ol-m, 12H), 2.17-2.23 (m, 2H), 4.04-4.13 (ol-m,2H), 4.40-4.48 (m, 1H), 6.04-6.36 (m, 1H), 6.95-7.23 (m, 1H) ppm.

EXAMPLE 9 Preparation of (SR)-N-Cbz-L-Leu-L-LeuN-[2-(phenylhexanol)]amide

To a stirred solution of N-BOC-L-Nle N-methoxy-N-methylamide (1.0 eq) inanhydrous THF at -78° C. under Ar is added slowly a solution of lithium4-fluorobenzene (3 eq) generated in situ. The reaction mixture isgradually warmed to R.T. After 3 h, 1 N HCl is added to the reactionmixture, and then extracted with EA. The combined organic layers arewashed with 1 N HCl, sat. NaHCO₃, sat. NaCl, dried (MgSO₄), filtered andconcentrated in vacuo to afford a crude residue. The residue is purifiedby flash chromatography on silica gel to give the ketone derivative.

The above intermediate (1.0 eq) is treated with 4 N HCl in dioxane atR.T. The reaction mixture is stirred for 1.5 h then concentrated invacuo. The solid is treated with anhydrous ether and concentrated invacuo to give 2S-amino-phenylhexanone hydrochloride.

To a stirred solution of 2S-amino-phenylhexanone hydrochloride (1.0 eq)in CH₂ Cl₂ at R.T. is added successively N-Cbz-L-Leu-L-Leu-OH (1.2eq),HOBT (2.2 eq) and EDC (1.1 eq). The reaction mixture is stirred for 18 hthen washed with sat. NaHCO₃, 1 N HCl (2×10 mL), sat. NaCl (2×10 mL),dried (MgSO₄), filtered and concentrated. Purification by flashchromatography on silica gel affords the peptide ketone intermediate.

To a stirred solution of the above intermediate (1.0 eq) in MeOH at R.T.is added NaBH₄ (1.0 eq). The reaction mixture is stirred for 3 h, thentaken up in EA washed with sat. NaHCO₃, 1 N HCl, sat. NaCl, dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyon silica gel affords the title compound.

EXAMPLE 10 Preparation of of (2SR)-(3S)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-butanol)]amide

To a stirred solution of N-BOC-L-Ala N-methoxy-N-methylamide (1.0 eq) inanhydrous THF at 0° C. under Ar is added lithium aluminum hydride(LiAlH₄) (1.0 eq). After 1 h the reaction mixture is treated with 1 NHCl then taken up in EA. The organic layer is washed with sat. NaHCO₃, 1N HCl, sat. NaCl, dried (MgSO₄), filtered and concentrated. Theresulting aldehyde is used in the next step without furtherpurification.

To a stirred solution of the above aldehyde (1.0 eq) in anhydrous THF at0° C. under Ar is added (trifluoromethyl)trimethylsilane (1.2 eq) inanhydrous THF followed by a catalytic amount of tetrabutylammoniumfluoride (TBAF). The resulting siloxy compound is hydrolyzed with 1 NHCl. The reaction mixture is taken up in EA. The organic layer is washedwith sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel givesthe N-BOC trifluoromethyl alcohol.

The above intermediate (1.0 eq) is treated with 4 N HCl in dioxane atR.T. The reaction mixture is stirred for 1.5 h then concentrated invacuo. The solid is treated with anhydrous ether and concentrated invacuo to give (2SR)-(3S)-3-amino-1,1,1-trifluoro-2-butanolhydrochloride.

To a stirred solution of (2SR)-(3S)-3-amino-1,1,1-trifluoro-2-butanolhydrochloride (1.0 eq) in CH₂ Cl₂ at R.T. is added successivelyN-Cbz-L-Leu-L-Leu-OH (1.2eq), HOBT (2.2 eq) and EDC (1.1 eq). Thereaction mixture is stirred for 18 h then washed with sat. NaHCO₃, 1 NHCl, sat. NaCl, dried (MgSO₄), filtered and concentrated. Purificationby flash chromatography on silica gel affords the title compound.

EXAMPLE 11 Preparation of (3SR)-(4S)-N-Cbz-L-Leu-L-Leu N-[(4-(ethyl2,2-difluoro-3-hydroxy-octanoate)]amide

To a stirred solution of N-BOC-L-Nle N-methoxy-N-methylamide (1.0 eq) inanhydrous THF at 0° C. under Ar is added LiAlH₄ (1.0 eq). After 1 h thereaction mixture is treated with 1 N HCl then taken up in EA. Theorganic layer is washed with sat. NaHCO₃, 1 N HCl, sat. NaCl, dried(MgSO₄), filtered and concentrated. The resulting aldehyde is used inthe next step without further purification.

To stirred solution of the freshly prepared above aldehyde (1.0 eq) andethyl bromodifluoroacetate (3.0 eq) in anhydrous THF at R.T. under Ar isadded Zn powder (4.0 eq). The reaction mixture is placed in a sonicationbath at R.T. for 1 h. The reaction is poured onto ice/H₂ O and theresulting slurry is filterd through celite, washing with ether. Theaqueous layer is separated and extracted with EA. The combined organiclayers are washed with sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄),filtered and concentrated. The crude residue is purified by flashchromatography on silcal gel to give the α,α-difluoro-β-hydroxy-ethylester product.

The above intermediate (1.0 eq) is treated with 4 N HCl in dioxane atR.T. The reaction mixture is stirred for 1.5 h then concentrated invacuo. The solid is treated with anhydrous ether and concentrated invacuo to give the hydrochloride adduct.

To a stirred solution of the above hydrochloride product (1.0 eq) in CH₂Cl₂ at R.T. is added successively N-Cbz-L-Leu-L-Leu-OH (1.2 eq), HOBT(2.2 eq) and EDC (1.1 eq). The reaction mixture is stirred for 18 h thenwashed with sat. NaHCO₃, 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyon silica gel affords the title compound.

EXAMPLE 12 Preparation ofN-BOC-(4S)-4-amino-1,1,1-trifluoro-2,2-difluoro-3-octanone

To a stirred solution of N-BOC-L-Nle N-methoxy-N-methylamide (1.0 eq) inanhydrous ether at -78°C. under Ar is added condensed pentafluoroethyliodide (CF₃ CF₂ I) (4.0 eq). To this mixture is addedmethyllithium-lithium bromide (CH₃ Li--LiBr) complex (4.0 eq) at a ratewhich maintains an internal reaction temperature below -65° C. Thereaction mixture is stirred for 2 h at -65 to -78° C., then poured ontoH₂ O. The aqueous phase is acidified with 1 N HCl (pH=3). The aqueousphase is extracted with ether. The combined organic layers are washedwith sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel affordsthe pentylfluoroethyl ketone, the title compound.

EXAMPLE 13 Preparation of (4S)-(3SR)-N-Cbz-L-Leu-L-LeuN-[4-(1,1,1-trifluoro-2,2-difluoro-3-octanol)]amide

N-BOC-(4S)-4-amino-1 ,1,1-trifluoro-2,2-difluoro-3-octanone, prepared inexample 12, is treated with 4 N HCl in dioxane at R.T. The reactionmixture is stirred for 1.5 h then concentrated in vacuo. The solid istreated with anhydrous ether and concentrated in vacuo to give thehydrochloride adduct.

To a stirred solution of the above hydrochloride product (1.0 eq) in CH₂Cl₂ at R.T. is added successively N-Cbz-L-Leu-L-Leu-OH (1.2 eq), HOBT(2.2 eq) and EDC (1.1 eq). The reaction mixture is stirred for 18 h thenwashed with sat. NaHCO₃, 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyon silica gel affords the pentafluoroethyl ketone peptide.

To a stirred solution of the above ketone (1.0 eq) in MeOH at R.T. isadded NaBH₄ (1.0 eq). The reaction mixture is stirred for 3 h, thentaken up in EA washed with sat. NaHCO₃, 1 N HCl, sat. NaCl, dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyon silica gel affords the title compound.

EXAMPLE 14 Preparation of (3SR)-(4S)-N-Cbz-L-Leu-L-LeuN-[4-(1,1,1-trifluoro-2,2-difluoro-3-methyl-3-octanol)]amide

To a stirred solution ofN-BOC-(4S)-4-amino-1,1,1-trifluoro-2,2-difluoro-3-octanone (1.0 eq),prepared in example 12, in anhydrous THF at -78° C. under Ar is added a3.0 M solution of methylmagnesium chloride in THF (2.2 eq). The mixtureis allowed to warm to R.T. After 1 h, the reaction mixture is treatedwith 1 N HCl and extracted with EA. The combined organic layers arewashed with sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel affordsthe desired tertiary alcohol.

The above tertiary alcohol (1.0 eq) is treated with 4 N HCl in dioxaneat R.T. The reaction mixture is stirred for 1.5 h then concentrated invacuo. The solid is treated with anhydrous ether and concentrated invacuo to give the hydrochloride adduct.

To a stirred solution of the above hydrochloride product (1.0 eq) in CH₂Cl₂ at R.T. is added successively N-Cbz-L-Leu-L-Leu-OH (1.2eq), HOBT(2.2 eq) and EDC (1.1 eq). The reaction mixture is stirred for 18 h thenwashed with sat. NaHCO₃, 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyon silica gel affords the the title compound.

EXAMPLE 15 Preparation (2SR)-(3SR)-N-Cbz-L-Leu-L-LeuN-[3-(1,1,1-trifluoro-2-methyl-2-heptanol)]amide

To a stirred solution of (2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol(1.0 eq) (as prepared in example 5) in anhydrous CH₂ Cl₂ (20 mL) at R.T.under Ar are added (BOC)₂ O (1.1 eq), DMAP (cat.) and Et₃ N (2.0 eq).After 1 h, the reaction mixture is washed with sat. NaHCO₃, 1 N HCl(2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel affordsthe the N-BOC protected compound.

To a stirred solution of the above product (1.0 eq) in 1:1 CH₂ Cl₂ :THFat R.T. under Ar is added trifluoroacetic acid (TFA) (3.0 eq) and theDess-Martin reagent (3.0 eq). The reaction mixture is stirred for 12 hand concentrated in vacuo. The resulting residue is treated with amixture of EA, sat. NaHCO₃ and saturated aqueous sodium thiosulfate(sat. Na₂ S₂ O₃). The organic layer is separated and washed with sat.NaHCO₃, sat. Na₂ S₂ 0₃, sat. NaCl, dried (MgSO₄), filtered andconcentrated. The residue is purifed by flash chromatography on silicagel to give the desired trifluoromethyl ketone.

To a stirred solution of the above trifluoromethyl ketone (1.0 eq) inanhydrous THF at -78° C. under Ar is added a 3.0 M solution ofmethylmagnesium chloride in THF (2.2 eq). The mixture is allowed to warmto R.T. After 1 h, the reaction mixture is treated with 1 N HCl andextracted with EA. The combined organic layers are washed with sat.NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel affords the desiredtertiary alcohol.

The above tertiary alcohol (1.0 eq) is treated with 4 N HCl in dioxaneat R.T. The reaction mixture is stirred for 1.5 h then concentrated invacuo. The solid is treated with anhydrous ether and concentrated invacuo to give the hydrochloride adduct.

To a stirred solution of the above hydrochloride product (1.0 eq) in CH₂Cl₂ at R.T. is added successively N-Cbz-L-Leu-L-Leu-OH (1.2eq), HOBT(2.2 eq) and EDC (1.1 eq). The reaction mixture is stirred for 18 h thenwashed with sat. NaHCO₃, 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyon silica gel affords the the title compound.

EXAMPLE 16 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-L-LeuN-[3-(1-(1'-phenyl-3'(trifluoromethyl)-pyrazoloxy)-2-heptanol)]amide

To the N-Cbz-L-Nle-CHN₂ (1.0 eq) is added HBr (g) and pyridine (5 mL).The mixture is taken up in EA and washed with 1 N HCl, sat. NaHCO₃, sat.NaCl, dried (MgSO₄), filtered and concentrated to give the bromomethylketone.

To a solution of the bromomethyl ketone (1.0 eq) in anhydrousdimethylformamide (DMF) under Ar at R.T. is added5-hydroxy-1-phenyl-3-(trifluoromethyl)-pyrazole (2.0 eq) and potassiumfluoride (2.0 eq). The reaction mixture is stirred at R.T. for 16 h, andextracted with EA. The combined organic layers are washed with sat.NaCl, sat. NaHCO₃, 1 N HCl, dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel affords thefurfurylthio-derivative. The derivative is treated with H₂ /Pd(C) inMeOH to give the free amine.

To a solution of the above free amine (1.0 eq) in anhydrous CH₂ Cl₂ atR.T. under Ar is added HOBT (2.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) andN-Cbz-L-Leu-L-Leu-OH (1.1 eq). After 6 h the reaction mixture is washedwith sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄) filtered andconcentrated. Purification by flash chromatography on silica gel givesthe ketone.

To a stirred solution of the ketone in methanol at 0° C. is added NaBH₄(1.0 eq). After 1 h the reaction mixture is warmed to R.T. The reactionmixture is stirred for 4 h, at R.T, then quenched by the addition of 1 NHCl (1 mL), concentrated, and extracted with EA. The combined organiclayers are washed with sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄),filtered and concentrated. Purification by flash chromatography onsilica gel gives the title alcohol.

EXAMPLE 17 Preparation of (2SR)-H-L-Leu N-[2-(ethyl4-methyl-4-pentenoate)]amide hydrochloride

To a stirred solution of ethyl 2-amino-4-methyl-4-pentenoatehydrochloride (as prepared in example 2) (0.70 g, 3.6 mmol) in CH₂ Cl₂(10 mL) at R.T. under Ar were added N-BOC-L-Leu-OH (1.0 9, 4.0 mmol),HOBT (1.19 g, 7.9 mmol), EDC (0.76 g, 4.0 mmol) and Et₃ N (0.55 mL, 4.0mmol). The reaction mixture was taken up in additional CH₂ Cl₂ (20 mL)and washed with sat. NaHCO₃ (2×10 mL), 1 N HCl (2×10 mL), sat. NaCl(2×10 mL), dried (MgSO₄), filtered and concentrated. The resultingresidue was purified by flash chromatography on silica gel (EA:H; 1:1)to afford the dipeptide ethyl ester as a white solid (1.05 g, 78.6%): ¹H NMR (CDCl₃, 300 MHz) δ0.92-0.97 (m, 6H), 1.25-1.30 (t, 3H, J=6 Hz),1.44-1.74 (m, 15H), 2.37-2.57 (m, 2H), 4.15-4.22 (m, 2H), 4.64-4.94 (m,4H) ppm.

To the above ester (1.0 g, 2.7 mmol) was added 4 N HCl/dioxane (15 mL),stirred at R.T. for 4 h, then the solvent was removed. Co-evaporationwith ether (3×5 mL) yielded the title compound as a white solid (0.8 g,96.3%): ¹ H NMR (CDCl₃, 300 MHz) δ0.89-1.00 (m, 6H), 1.21-1.31 (m, 3H),1.44-1.84 (m, 6H), 2.34 (m, 2H), 4.16-4.75 (m, 4H) ppm.

EXAMPLE 18 Preparation of(2SR)-N-[(2S)-2-benzoxy-4-methylpentanoyl1]-L-LeuN-(2-(4-methyl-4-pentenol)]amide

To a stirred solution of L-Leu-OH (5.0 9, 38.2 mmol) in 1 N H₂ SO₄ (50mL) at 0° C. was slowly added over 11/2 h a solution of sodium nitrite(NaNO₂ ) (7.5 g, 0.11 mmol) in water (20 mL) while maintaining thetemperature at 0° C. The reaction mixture was gradually warmed to R.T.,stirred for 24 h, and concentrated to give a white solid. The solid wasextracted with ether (5×50 mL). The combined organic layers were dried(MgSO₄), filtered and concentrated to give(2S)-2-hydroxy-4-methylpentanoic acid as an oil (4.1 g, 81.2%): ¹ H NMR(CDCl₃, 300 MHz) δ0.98 (d, 6H, J=12.0 Hz), 1.57-1.67 (m, 2H), 1.82-1.93(m, 1H), 4.36 (t, 1H, J=6.0 Hz) ppm.

To a stirred solution of the acid (4.0 9, 30.5 mmol) in anhydrous DMF(20 mL) at R.T. under Ar was added cesium carbonate (Cs₂ CO₃) (12.9 g,40.0 mmol) and methyl iodide (5.7 g, 40.0 mmol). The reaction mixturewas stirred for 16 h then taken up in EA (100 mL). The organic layer waswashed with sat. NaHCO₃ (3×20 mL), 1 N HCl (2×20 mL), dried (MgSO₄),filtered and concentrated. Purification by flash chromatography onsilica gel (EA:H; 1:4) gave methyl (2S)-2-hydroxy-4-methylpentanoate asa colorless oil (2.5 g, 57%): 1¹ H NMR (CDCl₃, 300 MHz) δ0.94-1.01 (m,6H), 1.56-1.74 (m, 2H), 1.87-1.96 (m, 1H), 3.79 (s, 3H), 4.24 (q, 1H,J=6.0 Hz) ppm.

To a stirred solution of methyl (2S)-2-hydroxy-4-methylpentanoate (0.5g, 3.4 mmol) in anhydrous CH₂ Cl₂ (10 mL) at R.T. under Ar was addedbenzyl 2,2,2-trichloroacetimidate (1.4 mL, 6.8 mmol) andtrifluoromethylsulfonic acid (25 μl). After 30 min the reaction mixturewas taken up in CH₂ Cl₂ (20 mL). The organic layer was washed with sat.NaCl (2×10 mL), 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated. Purification by flash chromatography onsilica gel (EA:H; 1:10) afforded methyl(2S)-2-benzoxy-4-methylpentanoate as an oil (0.6 g, 73.5%): ¹ H NMR(CDCl₃, 300 MHz) δ0.80-0.98 (m, 6H), 1.40-1.58 (m, 1H), 1.69-1.87 (m,2H), 3.74 (s, 3H), 3.93-4.06 (m, 1H), 4.42 (d, 1H, J=12.0 Hz), 4.68-4.80(m, 1H), 7.14-7.32 (m, 5H) ppm.

To the above methyl (2S)-2-benzoxy-4-methylpentanoate (0.69 g, 2.92mmol) in MeOH/H₂ O (5 mL/1 mL) was added LiOH.H₂ O (0.28 g, 11.7 mmol)and 30% H₂ O₂ (0.3 mL, 11.7 mmol). After stirring the reaction mixturefor 24 h, the mixture was treated with 1 N HCl (pH=3) and the methanolwas removed in vacuo. The aqueous layer was extracted with EA (4×15 mL).The combined organic layers were washed with 1 N HCl (2×10 mL), sat.NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.(2S)-2-benzoxy-4-methylpentanoic acid was isolated as a colorless oil(0.65 g, 100%): ¹ H NMR (CDCl₃, 300 MHz) δ0.82-1.60 (m, 6H), 1.53-1.62(m, 1H), 1.73-1.90 (m, 2H), 3.99-4.50 (m, 1H), 4.46 (d, 1H, J=12.0 Hz),4.72 (d, 1H, J=12.0 Hz) 7.10-7.26 (m, 5H) ppm.

To a solution of the product from example 17 (0.5 g, 1.63 mmol) inanhydrous CH₂ Cl₂ (10 mL) at R.T. under Ar was added the above acid (0.4g, 1.8 mL), HOBT (0.24 g, 1.8 mmol), EDC (0.35 g, 1.8 mmol) and Et₃ N(0.25 mL, 1.8 mmol). After 16 h the reaction mixture was washed withsat. NaHCO₃ (2×10 mL), 1 N HCl (2×10 ml), sat. NaCl (2×10 mL) dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyafforded the ethyl ester (0.5 g, 62.5%): Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.76 (t, 3H, J=6.0 Hz),0.91-0.99 (ol-m, 12H), 1.22-1.9 (ol-m, 9H), 2.05-2.18 (ol-m, 1H),2.34-2.43 (ol-m, 1H), 3.71-4.95 (ol-m, 9H), 7.20-7.38 (ol-m, 5H) ppm.

To a solution of the ester (0.5 g, 1.0 mmol) in anhydrous THF (10 mL) atR.T. under Ar was added LiBH₄ (0.02g, 1.0 mmol). Reaction mixture wasstirred for 4 h then quenched by the addition of 1 N HCl (1 mL),concentrated, and extracted with EA (3×10 mL). The combined organiclayers were washed with sat. NaHCO₃ (2×10 mL), 1 N HCl (2×10 mL), sat.NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated. Purificationby flash chromatography on silica gel (EA:H; 1:3) gave the title alcoholas an oil (0.13 g, 30%): Reporting a mixture of diastereomers ¹ H NMR(CDCl₃, 300 MHz) δ0.72-0.98 (ol-m, 2H), 1.10-2.21 (ol-m, 11H), 3.58-4.92(ol-m, 9H), 7.0-7.50 (ol-m, 7H) ppm.

EXAMPLE 19 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-LeuN-[3-(2-hydroxy-heptanoic acid)]amide

To a stirred solution of N-BOC-L-Nle N-methoxy-N-methylamide (1.0 eq) inanhydrous THF (10 mL) at 0° C. under Ar is added LiAlH₄ (1.0 eq), andstirred for 3 h at 0° C. followed by the addition of 1 N HCl (1 mL). Themixture is taken up in EA then washed with sat. NaHCO₃, 1 N HCl, sat.NaCl, dried (MgSO₄), filtered and concentrated to afford theN-BOC-L-norleucinal as a crude residue. To the residue is added anice-cold solution of NaHSO₃ (8 eq) and the mixture is stirred for 24 hat 5° C. To the resulting suspension is added EA and an aqueouspotassium cyanide solution (KCN) (8 eq). The reaction mixture is stirredat R.T. for 4 h. The organic phase is washed with water and concentratedto give the cyanohydrin.

The cyanohydrin is hydrolyzed in 4 N HCl/dioxane under reflux for 12 h.The solvent is removed and the residue is washed with anhydrous ether togive the hydrolyzate. To a stirred solution of N-Cbz-L-Leu-Leu-OH (1.0eq) in anhydrous CH₂ Cl₂ under Ar at R.T. is added CDl (1.1 eq). After30 min of stirring Et₃ N (2 eq) and the hydrolyzate (1.0 eq) are added.The mixture is stirred for 6 h, then concentrated. The residue istrituated with 1 N HCl washed with water and dried in vacuo to affordthe title compound.

EXAMPLE 20 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-L-Leu N-[3-(methyl2-hydroxy-heptanoate)]amide

To the product obtained from example 19 in anhydrous ether at 0° C. isadded diazomethane. After 3 h the solvent is removed and the residue ispurified by flash chromatography on silica gel to give the desiredproduct.

EXAMPLE 21 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-L-Leu N-[3-(benzyl2-hydroxyheptamide)]amide

To the product of example 19 in anhydrous CH₂ Cl₂ under Ar at R.T. isadded HOBT (1.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) and benzylamine (1.0eq). After 6 h the reaction mixture is washed with sat. NaHCO₃, sat. 1 NHCl, sat. NaCl, dried (MgSO₄), filtered and concentrated. The residue ispurified by flash chromatography on silica gel to afford the desiredproduct.

EXAMPLE 22 Preparation of (3SR)-(4S)-N-Cbz-L-Leu-L-Leu N-[4-(benzyl3-hydroxyoctamide)]amide

To a solution of N-BOC-L-norleucinal (1.0 eq), prepared by reducingN-BOC-L-Nle N-methoxy-N-methylamide as described in example 19, in THFat -78° C. under Ar is added ethyl lithioacetate (2.2 eq) prepared insitu by the addition of nBuLi (2.2 eq) to excess anhydrous ethylacetate. After 3 h, the reaction mixture is treated with 1 N HCl, andthe organic layer is washed with 1 N HCl, sat. NaHCO₃, sat. NaCl, dried(Mg SO₄), filtered and concentrated. Purification by flashchromatography on silica gel gives the ester.

The ester is treated with 4 N HCl/dioxane for 30 min, and concentratedin vacuo. The resulting solid is taken up in anhydrous ether andconcentrated in vacuo to give the hydrochloride. The hydrochloride isused without further purification in the next step.

To the hydrochloride (1.0 eq) in anhydrous CH₂ Cl₂ at R.T. under Ar isadded HOBT (2.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) and N-Cbz-L-Leu-Leu-OH(1.1 eq). After 6 h, the organic is washed with sat. NaHCO₃, 1 N HCl,dried (MgSO₄), filtered and concentrated. Purification by flashchromatography on silica gel gives the ester.

To a stirred solution of the above ester (1.0 eq) in MeOH/H₂ O is addedLiOH.H₂ O (2 eq) and H₂ O₂ (1.0 eq). After 4 h the reaction is quenchedby the addition of 1 N HCl and then extracted with EA (2×). The combinedorganic layers are washed with sat. NaCl, dried (MgSO₄) and concentratedto give the acid.

To a solution of the acid (1.0 eq) in anhydrous CH₂ Cl₂ at R.T. under Aris added EDC (1.0 eq), HOBT (1.0 eq), Et₃ N (1.0 eq) and benzylamine(1.1 eq). The reaction mixture is stirred for 3 h, washed with sat.NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄), filtered, concentrated andpurified by flash chromatography on silica gel affording the titlecompound.

EXAMPLE 23 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-L-LeuN-[3-(1-furfylthio-2-heptanol)]amide

To N-Cbz-L-Nle-CHN₂ (1.0 eq) is added HCl(g) and pyridine (5 mL). Themixture is taken up in EA and washed with 1 N HCl, sat. NaHCO₃, sat.NaCl, dried (MgSO₄), filtered and concentrated to give the chloromethylketone.

To a solution of the chloromethyl ketone (1.0 eq) in anhydrous THF underAr at R.T. is added furfuryl mercaptan (2.0 eq) and Et₃ N (2.0 eq). Thereaction mixture is stirred at R.T. for 16 h, and extracted with EA. Thecombined organic layers are washed with sat. NaCl, sat. NaHCO₃, 1 N HCl,dried (MgSO₄), filtered and concentrated. Purification by flashchromatography on silica gel affords the furfurylthio-derivative. Thederivative is treated with H₂ /Pd(C) in MeOH to give the free amine.

To a solution of the above amine (1.0 eq) in anhydrous CH₂ Cl₂ at R.T.under Ar is added HOBT (2.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) andN-Cbz-L-Leu-L-Leu-OH (1.1 eq). After 6 h the reaction mixture is washedwith sat. NaHCO₃, 1 N HCl, sat. NaCl, dried (MgSO₄) filtered andconcentrated. Purification by flash chromatography on silica gel givesthe ketone.

To a stirred solution of the ketone in methanol at 0° C. is added NaBH₄(1.0 eq). After 1 h the reaction mixture is warmed to R.T. The reactionmixture is stirred for 4 h, at R.T, then quenched by the addition of 1 NHCl (1 mL), concentrated, and extracted with EA (3×10 mL). The combinedorganic layers are washed with sat. NaHCO₃ (2×10 mL), 1 N HCl (2×10 mL),sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel (EA:H; 1:3) gave thetitle alcohol.

EXAMPLE 24 Preparation of(2SR)-N-[(2R)-[2-(1'-phenyl-1'-propene)-4-methylpentanloyl]]-L-LeuN-[2-(4-methyl-4-pentenol)]amide

To a stirred solution of 4-methylvaleric acid (10.8 mL, 86.1 mmol) inanhydrous CH₂ Cl₂ (25 mL) was added thionyl chloride (25 mL, 0.34 mmol).The mixture was placed under reflux for 24 h. Then solvent and excessthionyl chloride were removed in vacuo to give the acid chloride as anoil (10.2 g,90%). The acid chloride was used directly in the next step.

To the acid chloride (2.7 g, 20.3 mmol) in anhydrous CH₂ Cl₂ (50 mL) atR.T. under Ar was added DMAP (0.10 g), Et₃ N (4.6 mL, 33.8 mmol) and(4S,5R)-(-)-4-methyl-5-phenyl-2-oxazolidinone (3.0 g, 16.9 mmol). Thereaction mixture was stirred for 16 h then washed with 1 N HCl (2×10mL), sat. NaHCO₃ (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filteredand concentrated. Purification by flash chromatography on silica gel(EA:H; 1:20) afforded the imide as an oil (2.8 g, 61%): ¹ H NMR (CDCl₃,300 MHz) δ0.80-1.10 (m, 9H), 1.60-1.90 (m, 3H), 2.40-2.55 (m, 2H),4.80-4.90 (m, 1H), 5.70-5.80 (m, 1H), 7.20-7.50 (m, 5H) ppm.

To a solution of the imide (2.5 g, 9.14 mmol) in anhydrous THF (40 mL)at -78° C. under Ar was slowly added a 1.5 M solution of lithiumdiisopropylamide (LDA) in anhydrous THF (6.0 mL, 9.14 mmol) followed bycinnamyl bromide (1.8 g, 9.14 mL) in anhydrous THF (10 mL). The reactionmixture was stirred at -78° C. for 1 h then gradually warmed to R.T.Stirring was continued at R.T. for 1 h then the mixture was treated with1 N HCl (5 mL). The solvent was removed and the aqueous was taken up inEA (70 mL). The aqueous was separated and the organic was washed withsat. NaCl (2×10 mL), 1 N HCl (2×10 mL), sat. NaHCO₃ (2×10 mL), sat. NaCl(2×10 mL), dried (MgSO₄) filtered and concentrated. Purification byflash chromatography on silica gel (EA:H; 1:4) afforded the alkylatedderivative as an oil (3.5 g, 98.7%): ¹ H NMR (CDCl₃, 300 Mhz) δ0.73 (d,3H, J=6.0 Hz), 0.80-0.92 (m, 6H), 1.14-1.40 (m, 1H), 1.48-1.79 (m, 1H),1.75-1.84 (m, 1H), 2.42 (m, 2H), 4.10-4.20 (m, 1H), 4.71-4.78 (m, 1H),5.28 (d, 1H, J=9.0 Hz), 6.35 (m, 1H), 6.45 (m, 1H), 7.21-7.25 (m, 5H)ppm.

To a solution of the above product (0.65 g, 1.66 mmol) in MeOH/H₂ O(3:1) (20 mL) at R.T. was added LiOH.H₂ O (0.61 g, 4.98 mmol) and 30% H₂O₂ (0.83 mL). The reaction mixture was stirred for 4 h. The mixture wascooled to 0° C. and quenched by the addition of 1 M Na₂ S₂ O₃ (1.6 mL)and allowed to warm to R.T. After 14 h the resulting solution was pouredonto sat. NaHCO₃ (20 mL). The aqueous was extracted with CH₂ Cl₂ (3×30mL) then acidified with 1 N HCl (pH =3). The aqueous was then extractedwith CH₂ Cl₂ (3×20 mL) and the combined organics were dried (MgSO4),filtered and concentrated. The residue was purified by flashchromatography on silica gel (EA:H; 1:1) to give(2S)-2-(1'-phenyl-1'-propene)-4-methylpentanoic acid as an oil (0.30 g,78.0%): ¹ H NMR (CDCl₃, 300 MHz) δ0.88-1.01 (m, 6H), 1.29-1.42 (m, 3H),1.59-1.70 (m, 2H), 2.55 (m, 3H), 6.13 (m, 1H), 6.47 (d, 1H, J=6.0 Hz),7.31-7.40 (m, 5H) ppm.

To a stirred solution of L-Leu-OMe HCl (0.14 g, 0.70 mmol) and(2S)-2-(1'-phenyl-1'-propene)-4-methylpentanoic acid (0.16 g, 0.69 mmol)in anhydrous CH₂ Cl₂ (15 mL) at R.T. were added HOBT (0.19 g, 1.38mmol), EDC (0.15 g, 0.78 mmol) and Et₃ N (0.91 mL, 0.70 mmol). After 16h the reaction mixture was washed with sat. NaHCO₃ (2×10 mL), 1 N HCl(×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.Purification of the crude residue by flash chromatography on silica gel(EA:H; 1:1) afforded the methyl ester as an oil (0.24 g, 95%): ¹ H NMR(CDCl₃, 300 MHz) δ0.76-0.78 (d, 3H, J=6.0 Hz), 0.82-1.06 (m, 3H),1.26-1.41 (m, 1H), 1.44-1.72 (m, 2H), 2.22-2.30 (m, 2H), 2.35-2.46 (m,1H) 3.72 (s, 3H), 4.57 (m, 1H), 6.16 (m, 1H), 6.43 (m, 1H), 7.25-7.41(m, 5H) ppm.

To the above methyl ester (0.28 g, 0.78 mmol) in MeOH/H₂ O (15 mL-5 mL)with stirring at 0° C. was added 1 N LiOH.H₂ O (0.95 mL) and 30% H₂ O₂(1.4 mL). After 2 h 1 N HCl (4 mL) was added and the aqueous layer wasextracted with EA (3×30 mL). The combined organics were washed with sat.NaCl (2×20 mL), dried (MgSO₄), filtered and concentrated to give a cruderesidue. Purification by flash chromatography on silica gel (EA:H; 1:1)afforded the acid as an oil (0.21 g, 0.61 mmol): ¹ H NMR (CDCl₃, 300MHz) δ0.59-1.10 (m, 12H), 1.12-1.71 (m, 6H), 2.60-2.63 (m, 3H), 4.45 (m,1H), 6.21 (m, 1H) 6.40 (m, 1H), 7.25-7.41 (m, 5H) ppm.

To a stirred solution of the acid (0.25 g, 0.72 mmol) and ethyl2-amino-4-methyl-4pentenoate (0.16 g, 0.8 mmol) in anhydrous CH₂ Cl₂ (15mL) at R.T. were added HOBT (0.19 g, 1.44 mmol), EDC (0.15 g, 0.79 mmol)and Et₃ N (0.12 mL, 0.80 mmol). After 4 h the reaction mixture waswashed with sat. NaHCO₃ (2×10 mL), 1 N HCl (2×10 mL), sat. NaCl (2×10mL), dried (MgSO₄), filtered and concentrated. The residue was purifiedby flash chromatography on silica gel (EA:H; 1:1) to give(2SR)-[(2R)-[2-(1'-phenyl-1'-propene)-4-methyl pentanoyl]]-L-LeuN-[2-(ethyl 4-metyl-4-pentenoate)]amide as a solid (0.18 g, 51%): ¹ HNMR CDCl₃, 300 MHz) 60.81-1.10 (m, 12H), 1.30-1.73 (m, 15H), 2.27-2.51(m, 2H), 4.14-4.17 (m, 2H), 4.22-4.49 (m, 2H), 4.59 (m, 1H), 6.21 (m,1H), 6.43 (m, 1H), 7.17-7.19 9m, 2H), 7.25-7.41 (m, 5H) ppm.

To the above ethyl ester (0.3 g, 0.62 mmol) in anhydrous THF (20 mL) at0° C. under Ar with stirring was added LiBH₄ (47 mg, 2.15 mmol). After30 min at 0° C. the reaction mixture was warmed to R.T. Stirring wascontinued for 2 h then quenched with 1 N HCl (2mL). The mixture wasextracted with EA (3×10 mL). The combined organics were washed with sat.NaHCO₃ (2×10 mL), 1 N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated. Purification of the crude by flashchromatography on silica gel (EA) gave the alcohol as an oil (0.24 g,87%): ¹ H NMR (CDCl₃, 300 MHz) δ0.72-1.02 (m, 12H), 1.20-1.86 (m, 13H),2.21-2.56 (m, 4H), 3.60-3.68 (m, 2H), 4.21-4.25 (m, 1H), 4.80-4.85 (m1H), 6.10-6.15 (m, 1H), 6.45-6.50 (m, 1H), 7.17-7.19 (m, 2H), 7.25-7.35(m, 5H) ppm.

EXAMPLE 25 Preparation of (2SR)-N-Ac-L-Leu-L-LeuN[2-(trans-4-hexanol)]amide

The title compound was isolated as a white solid (1.4 g, 80.3%)following the procedure outlined in example 3: Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.89-0.97 (ol-m, 12H), 1.53-1.80(ol-m, 9H), 2.00-2.32 (ol-m, 5H), 3.49-3.90 (ol-m, 3H), 4.31-4.71 (ol-m,2H), 5.33-5.56 (ol-m, 2H), 6.95-8.10 (ol-m, 3H) ppm.

EXAMPLE 26 Preparation of (2SR)-N-Ac-L-Leu-L-LeuN-[2-(4-methyl-4-pentenol)]amide

The title compound was isolated as a white solid (0.9 g, 62.0%)following substantially the same procedure described in example 3:Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.86-0.91(ol-m, 12H), 1.58-1.72 (ol-m, 9H), 1.98-2.02 (d, 3H), 4.26-4.42 (ol-m,3H), 4.62-4.80 (ol-m, 2H), 6.00-6.05 (m, 1H), 6.70-6.82 (ol-m, 2H),9.53-9.55 (d, 1H) ppm.

EXAMPLE 27 Preparation of N-Dansyl-L-Leu-L-Leu-DL-norleucinol

The title compound was isolated as a pale yellow solid (0.13 g) usingthe methodology described in Example 3.

EXAMPLE 28 Preparation of N-Ac-L-Phe-L-Leu-DL-norleucinol

The title compound N-Ac-L-Phe-L-Leu-DL-norleucinol was obtained as awhite solid (0.19 g) following substantially the same proceduredescribed in Example 3.

EXAMPLE 29 Assays for Identification of Compounds Having Activity asModulators of the Processing of APP

A. Immunoblot assay for Aβ peptide

Human glioblastoma cells (ATCC Accession No. HTB16) were stablytransfected with a DNA expression vector encoding a 695 amino acidisoform variant of the amyloid precursor protein (APP) containing thefamilial Swedish double mutations at codons 670 and 671 (K to N and M toL, respectively; see Mullan et al. (1992) Nature Genet. 1:345-347) andan additional mutation at codon 717 (V to F; see Murrell et al. (1991)Science 254:97-99) to produce cells designated HGB 717/Swed. High levelsof Aβ are detectable in the conditioned medium isolated from HGB717/Swed cultured cells. The medium also contains larger secretedfragments, α-sAPP₆₉₅, which are alternatively processed APP fragmentswhose generation precludes Aβ formation.

HGB 717/Swed cells were grown at 37° C. under a 5% carbon dioxideatmosphere in Dulbecco's modified eagle medium (DMEM; Gibco)supplemented with 10% heat-inactivated fetal calf serum, 0.45% glucose,2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycinsulfate (Gemini Bioproducts). Approximately 1×10⁶ cells were incubatedovernight in 5 ml of DMEM containing varying μM final concentrations ofdesired test compounds or a DMSO control. Conditioned medium wascollected, and unwanted cells and debris were removed by sedimentationat 3,000 rpm at 4° C.

Aβ peptides were isolated from the medium by immunoaffinity purificationusing an Aβ-specific antibody. To reduce the interaction of non-specificbinding of unrelated proteins, such as serum proteins, to the Aβantibody, the medium was pre-treated with rabbit antisera and Protein ASepharose (Pharmacia) for 4 hours at 4° C. The sepharose-bound materialwas removed by centrifugation at 3,000 rpm at 4° C. for 10 min, and Aβpeptides were immunoaffinity purified from the clarified medium byincubation overnight with an affinity purified polyclonal rabbitantibody (referred to as 2939) prepared against a synthetic Aβ peptidecorresponding to amino acids 1 to 28. Protein A-conjugated sepharose wasadded to immobilize the Aβ-antibody complexes, and the resin waspelleted by centrifugation at 3,000 rpm at 4° C. for 10 min. TheAβ-antibody complexes were eluted from the matrix by denaturing thecomplex by boiling in the presence of SDS.

Equal portions of each sample were loaded on 16% Tricine gels (Novex),and subjected to electrophoresis. Resolved proteins were transferredfrom the gel to Hybond nitrocellulose (Amersham, Arlington Heights,Ill.) by electroblotting, and incubated with the commercially availablemonoclonal antibody 6E10 (obtained from Drs. Kim and Wisniewski,Institute for Basic Research, NY, see, published International PCTapplication WO 90/12871), which specifically recognizes Aβ residues 1 to17. Specifically bound antibody was detected using a biotinylated goatanti-mouse IgG secondary antibody (Sigma), followed by the addition ofstreptavidin conjugated to horseradish peroxidase (Amersham, ArlingtonHeights, Ill.), and documented by luminescent detection (Amersham).Levels of Aβ peptides were determined by laser densitometry ofvisualized films. A positive result in the assay is a decrease in theformation of the 4-kDa Aβ peptides relative to the DMSO control.Selected compounds provided herein were tested for and exhibitedactivity in this assay.

B. ELISA assay for total sAPP

Human glioblastoma cells (ATCC Acession No. HTB16) were stablytransfected with a DNA expression vector encoding the 695 amino acidisoform of the amyloid precursor protein (APP₆₉₅). The resulting cellsare designated HGB695 cells. High levels of secreted proteolyticprocessed fragments of APP₁₈₉₅ are detectable in the culture medium(sAPP₁₆₉₅).

Approximately 1×10⁵ cells were plated into 12-well dishes and were grownfor 72 hours at 37° C. under a 5% carbon dioxide atmosphere in 1 ml ofDulbecco modified eagle medium (DMEM) supplemented with 10%heat-inactivated fetal calf serum, 0.45% glucose, and 100 units/mlpenicillin, 100 μg/ml streptomycin sulfate and 2 mM L-glutamine.Following incubation, the medium was removed and 1 ml of supplementedDMEM medium containing 5 μl of DMSO or DMSO containing the desired testcompound within a range of about 5 to 100 μM (final concentration in thewell), was added to each well, and incubation was continued for 24hours. Unwanted cells and debris were removed by sedimentation at3,000×g for 10 min at room temperature. Supernatants were stored at -20°C. for analysis.

The capture monoclonal antibody P₂ -1, which recognizes an epitopelocated in the amino terminus of APP (see, e.g., U.S. Pat. No.5,270,165) was attached to the wells by incubating the antibody in theplate for 60 min at 37° C. The plates were washed three times with 0.3ml of 0.1% Tween-20 in phosphate-buffered saline (PBS). The non-specificinteraction of unrelated proteins (such as serum proteins that mayinterfere with the analysis) with the antibody was reduced by incubatingthe pre-treated wells for 30 min at 37° C. with a solution of 0.5%casein in PBS (150 μl/well). Wells were washed thoroughly with 0.1%Tween-20 in PBS prior to analysis of samples.

The conditioned medium supernatant was diluted 1:20 in 0.95 ml of 0.1%CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate) inPBS. Supernatant samples (100 μl/well) or sAPP standards (100 μl/well)of a range about 5 to 50 ng/ml were added to the pre-treated wells andincubated for 60 min at 37° C. The supernatant was removed and eachsample well was washed as described above. A horseradish peroxidase(HRP) conjugated goat affinity purified antibody, raised againstsAPP₆₉₅, was diluted in 0.1% Tween-20 in PBS and 10% goat serum andemployed as the "signal antibody". The unbound antibody was removed bywashing, and to each well, 0.1 ml of the chromagenic substrate K-BlueSolution (Elisa Technologies, Lexington, Ky.) was added and samples wereincubated for 15 min at ambient temperature. Reactions were stopped bythe addition of 0.1 ml of a 9.8% solution of phosphoric acid. Theoptical density of samples was measured by spectrophotometry at 450 nm.The concentration of sAPP₆₉₅ peptides in the conditioned medium wasestimated from the sAPP₆₉₅ standard curve. Samples were analyzed induplicate with the appropriate standards and reference controls [i.e., aknown drug, such as N-acetylleucylleucylnorleucinal of given potency andconcentration].

C. Cell lysate assay

In this assay, the effect of compounds on the modulation of thegeneration of partially processed C-terminal Aβ-containing amyloidogenicpeptides is examined. HGB695 human glioblastoma cells were employed andgrown in 12-well dishes essentially as described in Example 29B with thefollowing modifications. The DMEM growth media were supplemented withvarying μM concentrations of compounds or DMSO control and 100 μMleupeptin and 1 μM PMA phorbol ester and were incubated with cellcultures for 16 hours and cells were grown to approximately 2.5×10⁶cells per well.

Harvested cells from each well were lysed in 100 μl of lysis buffercontaining 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% NP-40, 0.1% SDS and0.5% deoxycholate supplemented with 1 mM PMSF. Equal volumes of celllysates in Laemmli SDS buffer were loaded onto 16% SDS-Tricinepolyacrylamide gels (Novex) and subjected to electrophoresis. Separatedproteins were transferred to supported nitrocellulose (BioRad) byelectroblotting. Nonspecific binding of proteins to the nitrocellulosemembrane was blocked by incubating in a solution of 5% non-fat driedmilk in PBS. The nitrocellulose membrane was washed three times in PBSand then incubated in PBS containing a 1:5000 dilution of a rabbitpolyclonal antibody raised against the C-terminal 19 amino acids of APP(provided by S. Gandy, Rockefeller University, NY). The nitrocellulosemembrane was washed as described above and incubated with a secondarybiotinylated goat anti-rabbit IgG antibody. Specifically bound antibodywas detected using a streptavidin horseradish peroxidase conjugate, andvisualized in combination with an enhanced chemoluminescent detectionkit (Amersham). Potentially amyloidogenic peptides greater than 9 andless than 22 kDa were quantitated by densitometric scans of developedfilms within the linear range as described in Example 29B. A positiveresult for a compound in the cell lysate assay is denoted by a decreasein the levels of the protein bands greater than 9 and less than 22 kDarelative to the appropriate control samples.

D. ELISA assay for α-sAPP

Human HGB695 glioblastoma cells transfected with DNA encoding the 695amino acid isoform of APP were grown and treated with test compound orDMSO as described in Example 29B. Medium from the cultured cells wasalso obtained as described in Example 29B and analyzed for α-sAPP in anELISA assay as follows. The wells of a 96-well microtiter plate werecoated with a monoclonal antibody that specifically recognizes the aminoterminus of human sAPP (e.g., monoclonal antibody P₂ -1) by incubatingthe antibody in the plate for 60 min at 37° C. The plates were washedthree times with 0.3 ml of 0.1% Tween-20 in PBS. The non-specificinteraction of unrelated proteins (e.g., serum peptides that mayinterfere with the analysis) with the antibody was reduced by incubatingthe pre-treated wells with a solution of 0.5% casein in PBS for 30 minat 37° C. Wells were washed with 0.1% Tween-20 in PBS prior to analysisof media samples.

The conditioned media were diluted 1:20 in 0.95 ml of 0.1% CHAPS in PBS.Media samples (100 μl/well) or α-sAPP standards (100 μl/well) in a rangeof about 3 to 18 ng/ml were added to the wells for a 60 min incubationat 37° C. The solution was then removed and each sample well was washedas described above. A horseradish peroxidase-conjugated rabbit affinitypurified antibody raised against a synthetic peptide consisting of thefirst 15 amino acids of Aβ (referred to as antibody 3369) was diluted in0.1% Tween-20 in PBS plus 10% normal rabbit serum and added to the wellsas the signal antibody. The plates were incubated for 60 min at 37° C.and then washed to remove unbound antibody. The chromogenic substrateK-Blue Solution (Elisa Technologies, Lexington, Ky.) was added to thewells and allowed to incubate for 15 min at ambient temperature. Thereactions were stopped by addition of 0.1 ml of a 9.8% solution ofphosphoric acid. The optical density of the samples was measured byspectrophotometry at 450 nm.

The concentration of α-sAPP in the media was estimated from the α-sAPPstandard curve. Samples were analyzed in duplicate.

EXAMPLE 30 Method of Indicating Alzheimer's Disease

Total s-APP and α-sAPP levels in cerebrospinal fluid (CSF) of normalsubjects and members of a Swedish family carrying mutations of the APPgene at codons 670 and 671 (APP_(670/671)) were measured and compared.The APP_(670/671) mutation in the Swedish family is associated with ahigh incidence of early onset Alzheimer's disease (AD). The clinicaldiagnosis of AD in the Swedish family harboring the mutation was basedon NINCDS-ADR_(D) A criteria [McKhann, et al. (1984) Neurology34:939-9441. The diagnosis was confirmed by neuropathologic examinationof the brain of one deceased mutation carrier [Lannfelt, et al. (1994)Neurosci. Lett. 168:254-256]. Cognitive functioning was assessed withthe Mini Mental State Examination (MMSE) [Folstein, et al. (1975) J.Psychiatry Res. 12:189-198]. The presence or absence of theAPP_(670/671) mutation was determined by polymerase chain reaction (PCR)nucleic acid amplification and restriction enzyme digestion according toa previously established procedure [Lannfelt, et al. (1993) Neurosci.Lett. 153:85-87].

Lumbar CSF was obtained from eight normal non-carriers in the family,two presymptomatic healthy mutation carriers, and four mutation carriersclinically symptomatic for AD. CSF samples were placed on ice, aliquotedand stored at -20° C. until tested.

A. Measurement of APP Levels

Total sAPP and α-sAPP levels in the CSF samples were quantitated using asandwich enzyme-linked immunosorbent assay (ELISA) and immunoblottingfollowed by laser-scanning densitometry, respectively.

Standards used in the assays were obtained by isolation of total sAPPand α-sAPP from medium conditioned by human neuroblastoma IMR₃₂ cells[ATCC Accession No. CCL127] or the HGB695 cells, described above inExample 29B, as follows. Conditioned medium was filtered to remove largecell debris, and sAPP was extracted by passing the medium over an anionexchange column using Toyopearl DEAE 650C resin (Toso-Hass,Philadelphia, Pa.). The bound sAPP was eluted from the column using alinear gradient of 0 to 0.6 M NaCl in 50 mM sodium phosphate, pH 7.5.All sAPP-containing eluate fractions were pooled and loaded onto animmunoaffinity column containing a monoclonal antibody that specificallyrecognizes an amino-terminal epitope of human APP (for example,monoclonal antibody P₂ -1 raised against native human PN-2) [see, e.g.,U.S. Pat. No. 5,213,962] linked to Toyopearl AF-Tresyl 650 M resin(Toso-Hass). Bound SAPP was eluted from the column with 0.1 M sodiumcitrate, pH 2.0. To separate α-sAPP from the other soluble forms of sAPPcontained in total sAPP that do not contain at least the amino-terminalportion of Aβ, the total sAPP was loaded onto a Sepharose 4Bimmunoaffinity adsorption column containing a monoclonal antibody thatrecognizes an epitope within the first ˜17 amino acids of Aβ (forexample, monoclonal antibody 6E10). Specifically bound α-sAPP was elutedfrom the column with 0.1 M sodium citrate, pH 3.0. The solution pH ofthe purified sAPPs was adjusted to 7.2 and 1-ml aliquots were stored at-70° C.

B. Quantitation of Total sAPP

The ELISA used to quantitate total sAPP levels in CSF samples employed amonoclonal antibody, such as P₂ -1, discussed above, that specificallyrecognizes an amino-terminal epitope of human APP as the captureantibody. The capture antibody was attached to the wells of a 96-wellmicrotiter plate by incubating the plate with the antibody (that hadbeen diluted in PBS, pH 7.2) for 60 min at 37° C. The plates were thenwashed three times with 0.3 ml of 0.1% Tween-20 in PBS. The wells werealso incubated with a solution of 0.5% casein in PBS (150 μl/well) for30 min. at 37° C.

CSF samples (100 μl diluted 1:20) or sAPP standards (100 μl) containinga range of 5 to 50 ng/ml were added to the wells and allowed to incubatefor 60 min at 37° C. Following incubation, the wells were washedthoroughly with 0.1% Tween-20 in PBS. A goat anti-human APP polyclonalantibody raised against immunopurified APP from medium conditioned bycultured IMR₃₂ human neuroblastoma cells (American Type CultureCollection Accession No. 127) conjugated to horseradish peroxidase wasused as the signal antibody. The antibody was diluted 1:500 in PBS and10% normal goat serum, pH 7.2, containing 0.1% Tween-20, added to thewells, and incubated for 60 min at 37° C. Unbound antibody was removedby washing as described above. To detect the bound antibody, 0.1 ml ofthe chromogenic substrate K-Blue Solution (Elisa Technologies,Lexington, Ky.) was added to the wells and allowed to incubate for 15min at ambient temperature. Reactions were stopped by addition of 0.1 mlof a 9.8% solution of phosphoric acid. The optical density of thesamples was measured by spectrophotometry at 450 nm. The concentrationof sAPP peptides in the CSF sample was estimated from the standardcurve. Samples were analyzed in duplicate.

Total sAPP levels were also measured using quantitative immunoblottingessentially as described below for measurement of α-sAPP, except using amonoclonal antibody raised against a recombinant APP-containing fusionprotein (e.g., 22C11 available from Boehringer Mannheim, Indianapolis,Ind.) at a concentration of 0.3 μg/ml to specifically detect sAPP andusing purified sAPP as a standard. Quantification of total sAPP byquantitative immunoblot gave a 95% correlation to quantification byELISA.

C. Quantitation of α-sAPP

For immunoblot assays of α-sAPP contained in the CSF samples, 5-10 μl ofsample and purified standard α-sAPP of known concentrations wereanalyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) under reducing conditions. Samples were diluted into Laemmlisample buffer and loaded onto 7.5% SDS-PAGE gel. After separation, theproteins were transferred to polyvinylidene difluoride membranes (PVDFImmobilon, Millipore, Bedford, Mass.) in CAPS transfer buffer (5 mM3-[cyclohexylamine]-1-propanesulfonic acid, pH 11.0, 5% (v/v) methanol).Nonspecific binding of protein to membranes was blocked with PBScontaining 5% (w/v) non-fat dried milk and then incubated for 1 hr witha monoclonal antibody (20ml of 0.2 μg/ml) directed against theamino-terminus of Aβ (e.g., 6E10), and washed three times for one mineach time in 20 ml of PBS and 0.1% Tween. Specifically bound antibodywas detected using a biotinylated goat anti-mouse secondary antibody(Sigma) and a streptavidin-peroxidase conjugate (Amersham, ArlingtonHeights, Ill.) in combination with an enhanced chemiluminescencedetection system (Amersham, Arlington Heights, Ill.). The blots wereexposed to Kodak Scientific Imaging film X-OMAT AR and developed using aKodak X-OMAT developer. Quantitation of the α-sAPP protein in the blotswas performed by laser-scanning densitometry. Developed films within thelinear range (or multiple exposures) were scanned at 50 μM pixel sizeusing a densitometer (Molecular Dynamics, Sunnyvale, Calif.), and thedata were quantified using the ImageQuaNT software system (MolecularDynamics). Quantified volumes of α-sAPP standard were used to generatestandard curves. From the standard curves, the levels of α-sAPP in ng/mlwere determined.

D. Comparison of sAPP and α-sAPP Levels in CSF of Normal Subjects andMutation Carriers

Assays of sAPP and α-sAPP levels in CSF from normal subjects and Swedishmutation carriers were performed. Mann-Whitney non-parametric statisticswere used for comparison of the data from the experimental groups.Correlations were investigated with Pearson's and Spearman's rankcorrelation coefficients. Significance levels were set at p<0.05. TheCSF of diseased carriers had lower levels of α-sAPP than the CSF samplesof non-carriers, with no overlap between the two groups (z=-2.72;p=0.007). The CSF obtained from the four AD subjects had lower levels ofα-sAPP than that of the two pre-symptomatic AD carriers. There was astrong inverse correlation between α-sAPP concentration and age in themutation carriers (R=0.94; p=0.005). In the mutation carriers, ˜25% ofthe total sAPP in CSF was α-sAPP compared to 33% in CSF of non-carriers.This was a statistically significant difference.

The results indicate that α-sAPP and the ratio of α-sAPP to total sAPPin CSF are useful markers in the detection of neurodegenerativedisorders characterized by cerebral deposition of amyloid (e.g., AD) andin monitoring the progression of such disease. Furthermore, this assaysystem can be used in monitoring therapeutic intervention of thesediseases.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

We claim:
 1. A compound having the formula (II) or (III): ##STR8## or ahydrate, isostere, stereoisomer or mixture thereof, or apharmaceutically acceptable salt thereof, but with the proviso that: (1)at least one of R₁ and R₃ is not a side chain of a naturally-occurringamino acid; and (2) when R₁ is the side chain from a non-naturallyoccurring amino acid, R₁ is not the side chain of cyclohexylalanine orcyclohexylglycine,wherein X is --CH(OH)CF₃, --CH(OH)C₂ F₅, --(CH₂)_(r)CH(OH)C_(k) H.sub.(2k+1-s) F_(s) in which k is 1-6 and s is 0 to 2k+1,or --CH(OH)C₆ H.sub.(5-q) F_(q) in which q is 0 to 5; r is 0-5; thealkyl and aryl portions of X are unsubstituted or are substituted withone or more substituents independently selected from G; G is halogen,lower alkyl, alkoxy, haloalkyl, NO₂, nitrile, S-alkyl, phenyl, or --NRR;R is H, alkyl, OH or halo-lower alkyl; the heterocyclic rings containone or two heteroatoms; R₁, R₂, R₃, R₄, R₈, R_(A), R_(B), Q and n areselected from among (i), (ii) or (iii) as follows: (i) R₁, R₃ and R_(B)are each independently selected from the group consisting of a sidechain of a naturally occurring α-amino acid, H, alkyl, alkenyl, alkynyl,aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl,heteroaralkenyl, Y-substituted aryl, aralkyl, aralkenyl, aralkynyl, andZ-substituted heteroaryl, heteroaralkyl, heteroaralkenyl, in which Y isselected from the group consisting of halogen, lower alkyl, alkoxy, OH,haloalkyl, nitrile, S-alkyl, phenyl, and --NRR, R is H, alkyl, loweralkyl, OH or halo-lower alkyl, and Z is lower alkyl or halo loweralkyl;R₂, R₄ and R₈ are each independently selected from H and loweralkyl; Q is selected from the group consisting of --C(O)--, --O--C(O),--S(O)₂ -- and HN--C(O)--; n is zero or one; R_(A) is --(T)_(m)--(D)_(m) --R₁ in which T is O, or NH, D is C₁₋₄ alkyl or C₂₋₄ alkeneand m is zero or one; or (ii) R₃, R₄, R_(A), R_(A), R_(B), Y, Q, and nare as defined in any of (i) or (iii),R₈ is H; and R₁ and R₂ are eachindependently selected as follows:(a) from lower alkyl; lower alkylbearing a heteroatom that is substituted with one of the followinggroups; lower alkyl, alkoxy, hydroxyl, haloalkyl, nitrile and phenyl;and a heteroatom that is substituted with one of the following groups;lower alkyl, alkoxy, hydroxy, haloalkyl, nitrile and phenyl; with theproviso when more than one heteroatom is present, there is at least onecarbon atom between each heteroatom, and (b) R₁ and R₂ are unsubstitutedor substituted with Y, and (c) together with the atoms to which they areattached form a 4-6 membered heterocyclic moiety; or (iii) R₁, R₂, R₈,R_(A), R_(B), Y, Q and n are as defined in any of (i) or (ii);R₃ and R₄are each independently selected as follows:(a) from lower alkyl: loweralkyl bearing a heteroatom that is substituted with one of the followinggroups; lower alkyl, alkoxy, hydroxyl, haloalkyl, nitrile and phenyl;and a heteroatom that is substituted with one of the following groups;lower alkyl, alkoxy, hydroxy, haloalkyl, nitrile and phenyl; with theproviso when more than one heteroatom is present, there is at least onecarbon atom between each heteroatom, and (b) R₃ and R₄ are unsubstitutedor substituted with Y, and (c) together with the atoms to which they areattached form a 4-6 membered heterocyclic moiety; and the resultingcompound modulates the processing of amyloid precursor protein (APP). 2.A compound of claim 1, wherein:R₁ is selected from the group consistingof n-butyl, 2-methylpropenyl and 2-butenyl; R₂, R₄ and R₈ are eachindependently selected from the group consisting of hydrogen, methyl andethyl; R₃ is iso-butyl; and X is selected from the group consisting of--(CH₂)_(r) CH(OH)CF₃, --(CH₂)_(r) CH(OH)C_(k) H.sub.(2k+1-s) F_(s) and--CH(OH)CF₃.
 3. A compound of claim 2 that corresponds to formula II. 4.A compound of claim 3; wherein X taken together with the carbon atom towhich it is bonded is a secondary trifluoromethyl alcohol.
 5. A compoundof claim 2, wherein X taken together with the carbon atom to which it isbonded is a secondary trifluoromethyl alcohol.
 6. A compound of claim 1,wherein X taken together with the carbon atom to which it is bonded is asecondary trifluoromethyl alcohol.
 7. A compound of claim 1 thatcorresponds to formula II.
 8. A compound of claim 7, wherein X takentogether with the carbon atom to which it is bonded is a secondarytrifluoromethyl alcohol.
 9. A pharmaceutical composition, comprising atherapeutically effective amount of a compound of claim 1 in aphysiologically acceptable carrier.
 10. A pharmaceutical compositionformulated for single dosage administration, comprising, in aphysiologically acceptable carrier, a therapeutically effective amountof a compound of claim
 1. 11. A compound that is selected from the groupconsisting of(2SR)-(3SR)-N-Cbz-L-Leu-N-[3-(1,1,1,-trifluoro-2-heptanol)]amide and(2SR)-(3SR)-N-valeroyl-L-Leu-N-[3-(1,1,1,-trifluoro-2-heptanol)]amide.12. A compound of claim 11 that is(2S)-(3S)-N-Cbz-L-Leu-N-[3-(1,1,1-trifluoro-2-heptanol)]amide.
 13. Acompound of claim 11 that is(2R)-(3R)-N-Cbz-L-Leu-N-[3-(1,1,1-trifluoro-2-heptanol)]amide.
 14. Acompound of claim 11 that is(2S)-(3R)-N-Cbz-L-Leu-N-[3-(1,1,1-trifluoro-2-heptanol)]amide.
 15. Acompound of claim 11 that is(2R)-(3S)-N-Cbz-L-Leu-N-1[3-(1,1,1-trifluoro-2-heptanol)]amide.
 16. Amixture of diastereomers, comprising(2SR)-(3SR)-N-Cbz-L-Leu-N-[3-(1,1,1-trifluoro-2-heptanol)]amide.
 17. Acompound selected from the group consisting of(2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol, (2SR)-(3SR)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3SR)-N-valeroyl-L-LeuN-[3-(1,1,1-trifluoro-2-heptanol)]amide, (2SR)-(3S)-N-Cbz-L-LeuN-[3-(1,1,1-trifluoro-2-butanol)]amide,(2SR)-N-[(2S)-2-benzoxy-4-methylpentanloyl]-L-LeuN-[2-(4-methyl-4-pentenol)]amide and(2SR)-N-[(2R)-[2-(1'-phenyl-1'-propene)-4-methylpentanoyl]]-L-LeuN-[2-(4-methyl-4-pentenol)]amide.